4-O-(aminoglycosyl)- or 4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamine derivatives

ABSTRACT

As new compounds are now synthetized 5-deoxy-5,5-difluoro derivatives of aminoglycosidic antibiotics of neamine, kanamycin A-series, kanamycin B-series, gentamicin-series and seldomycin-series. Further, the 1-N-(α-hydroxy-ω-aminoalkanoyl) derivatives are synthetized from 5-deoxy-5,5-difluorokanamycins A and B, as well as their analogues now obtained. The 5-deoxy-5,5-difluoro derivatives of the aminoglycosidic antibiotics now provided according to this invention have such antibacterial activities enhanced much more than or equal to those of the respectively corresponding parent aminoglycosidic antibiotics, while these 5-deoxy-5,5-difluoro derivatives exhibit values of the 50% lethal dosage (LD 50 ) which are improved remarkably better when they are intravenously administered to mice, so that they have remarkedly reduced toxicities to mammals.

TECHNICAL FIELD

This invention relates to new semi-synthetic aminoglycosidicantibiotics, namely such 5-deoxy-5,5-difluorostreptamine derivativeswhich are derived from aminoglycosidic antibiotics or their deoxyderivatives, as well as 1-N-acylated derivatives of said5-deoxy-5,5-difluorostreptamine derivatives. This invention also relatesto antibacterial compositions containing these new compounds. Further,this invention relates to processes for the production of these newcompounds. These new compounds of this invention are useful asantibacterial agent for therapeutic treatment of bacterial infections,since they exhibit high antibacterial activities against such bacteriasensitive to aminoglycosidic antibiotics, and also against such bacteriaresistant to aminoglycosidic antibiotics and have a characteristicallyand remarkably reduced toxicity.

BACKGROUND ART

Various deoxy derivatives of kanamycin A, B or C have usefulantibacterial activities, but the antibacterial spectra of these knowndeoxy-derivatives of kanamycins are of different ranges. Accordingly, itis always demanded that new, antibacterial compounds having any moreexcellent properties than the known kanamycin derivatives should beproduced and provided.

The present inventors have synthetized 3'-deoxy-3'-fluorokanamycin A(see Japanese patent application first publication "Kokai" No. 40297/86and U.S. Pat. No. 4,634,688). Further, the present inventors havesynthetized 3'-deoxy-3'-fluorokanamycin B and found that3'-deoxy-3'-fluorokanamycin B has remarkable antibacterial activitiesagainst various gram-positive and gram-negative bacteria, including theresistant bacteria (see Japanese patent application first publication"Kokai" No. 140597/86). And, the present inventors have synthetized3',4'-dideoxy-3'-fluorokanamycin B (see Japanese patent applicationfirst publication "Kokai" No. 51694/87 and U.S. Pat. No. 4,845,082specification).

Furthermore, the present inventors have synthetized 1-N-[(RS)- or(S)-3-amino-2-hydroxy-propionyl]- or1-N-[(S)-4-amino-2-hydroxybutyryl]-3'-deoxy-3'-fluorokanamycin A or B(see Japanese patent application first publication "Koaki" No.236791/86).

Moreover, the present inventors have synthetized 2',3'-dideoxy-2'-fluorokanamycin A (see the specification of Japanesepatent application first publication "Kokai" No. 143393/86) as well as1-N-[(RS)- or (S)-3-amino-2-hydroxypropionyl]- or1-N-[(S)-4-amino-2-hydroxybutyryl]-2',3'-dideoxy-2'-fluorokanamycin A(see Japanese patent application first publication "Koaki" No. 93296/87and U.S. Pat. No. 4,661,474specification).

On the other hand, a literature "Aminocyclitol Antibiotics" pages371-392, edited by K. L. Rinehart and T. Suami (published from AmericanChemical Society in 1980) discloses the production of5-deoxy-5-fluoro-5-epi-sisomicin according to a method comprisingreacting diethylaminosulfur trifluoride (hereinafter abbreviated asDAST) with such a protected derivative of sisomicin whose all the aminogroups and all the hydroxyl groups except the 5-hydroxyl group have beenprotected. Further, a literature "Journal of Carbohydrate Chemistry"Vol. 1, page 289 (1982) discloses the production of4"-deoxy-4"-fluoro-4"-epi-kanamycin A with starting from kanamycin A,although this literature shows that the antibacterial activity of this4"-deoxy-4"-fluoro-4"-epi-kanamycin A is lower than that of kanamycin A.

Furthermore, a literature "Tetrahedron Letters" Vol. 24, No. 17, pages1763-1766(1983) discloses that 6"-deoxy-6"-fluorokanamycin A isproduced, that 5,6"-dideoxy-5,6"-difluoro-5-epi-kanamycin A is producedand further that 5-deoxy-5-fluoro-5,4"-di-epi-kanamycin A is produced.

In this literature "Tetrahedron Letters" Vol. 24, No. 17, pages1763-1766(1983), there is disclosed that 6"-deoxy-6"-fluorokanamycin A,5,6"-dideoxy-5,6"-difluoro-5-epi-kanamycin A and5-deoxy-5-fluoro-5,4"-di-epi-kanamycin A show antibacterial activitiessubstantially as high as that of kanamycin A. Accordingly, thisliterature does not give any teaching that the antibacterial activity ofkanamycin A can be enhanced by replacement of the 6"-hydroxyl groupand/or the 5-hydroxyl group of kanamycin A by a fluorine atom. In thisliterature, there is not given any reference to toxicity and otherbiological activities of the fluorokanamycin A derivatives disclosedtherein.

In another literature "Chemical Abstracts" 90, 104, 301 (1979), there isdisclosed a method of producing 5-deoxy-5-fluoro-5-epi-kanamycin A,which comprises reacting DAST with a protected derivative of kanamycin Awhose the amino groups and the hydroxyl groups other than the 5-hydroxylgroup have been protected with conventional protective groups. But, thisliterature does not disclose that antibacterial activity of5-deoxy-5-fluoro-5-epi-kanamycin A so obtained is improved over that ofkanamycin A.

As will be clear from the above, hithertobefore it has not been foundthat the antibacterial activity of sisomicin can advantageously beimproved with the cases of 5-deoxy-5-fluoro-5-epi-sisomicin and5-deoxy-5-fluoro-sisomicin which are obtained by reacting the 5-hydroxylgroup with DAST and thereby replacing the 5-hydroxyl group by a fluorineatom, with or without possible concomitant inversion of the stericarrangement of the 5-substituent. Besides, it has not yet beenrecognized that the antibacterial activity of kanamycin A canadvantageously be enhanced with the case of the5-deoxy-5-fluoro-5-epi-kanamycin A, as compared to the parent kanamycinA.

Thus, the present inventors have already found that3'-deoxy-3'-fluorokanamycin A or B, 3',4'-dideoxy-3'-fluorokanamycin Band 2',3'-dideoxy-2'-fluorokanamycin A can each exhibit their improvedantibacterial activities, as compared to those of their correspondingparent compounds not having the fluoro substituent. On the other hand,the present inventors have recognized that the replacemnt of the5-hydroxyl group of sisomicin or kanamycin A by the fluoro substituentcannot bring about any advantageous improvement or enhancement in theantibacterial activity of the parent sisomicin or kanamycin A. Despitingthis recognition, the present inventors have studied to synthetize5-deoxy-5-fluorokanamycin B. In addition to our success to synthetize5-deoxy-5-fluorokanamycin B, the present inventors have succeeded insynthetizing 5,3'-dideoxy-5-fluorokanamycin B from 3'-deoxy-kanamycin B(namely, tobramycin); 5,4'-dideoxy-5-fluorokanamycin B from4'-deoxykanamycin B (see the "Bull. Chem. Soc. Jpn." 50, page 2362(1977)); and 5,3',4'-trideoxy-5-fluorokanamycin B from3',4'-dideoxykanamycin B (namely, dibekacin) (see the specification ofJapanese patent application first publication "Kokai" No. 39891/88 andEuropean patent application publication No. 0 259 014 A2).

Hithertobefore, the aminoglycosidic antibiotics have been used inclinics to exhibit such excellent curative effects in the therapeutictreatment of bacterial infections that are not obtainable with the otherkinds of antibiotics, because the aminoglycosidic antibiotics aresoluble in water and hence are well systemic to the tissues of humanbody. By virtue of this, the aminoglycosidic antibiotics are frequentlyutilized in the therapeutic treatment of patients sufferring from heavybacterial infections for which the other kinds of antibiotics are noteffective. In this therapeutic treatment with the aminoglycosidicantibiotics, however, intravenous injection of aqueous solutions of theaminoglycosidic antibiotics is usually and mainly made, so that theacute toxicity of the aminoglycosidic antibiotics is rapid to develop inthe patients as treated, often resulting in that dosage of saidantibiotics must be reduced and/or administration of said antibioticsmust be interrupted. In recent years, therefore, the doctors inhospitals are likely to have an idea that the aminoglycosidicantibiotics are of high toxicity. Thus, there prevails actually such atendency that the doctors would have a fear to the toxicity of theaminoglycosidic antibiotics and rather refrain from administering theaminoblycosidic antibiotics to such patients which should need to betherapeutically treated by administration of the aminoglycosidicantibiotics.

The present inventors have made efforts for 10 years or more in the pastto solve the problem that the toxicity of the aminoglycosidicantibiotics is reduced. During recent investigations, the presentinventors incidentally have discovered that the above-mentioned5-deoxy-5-fluorokanamycin B now synthetized and its analogues show amore or less reduced toxicity (LD₅₀ value of ca. 135 mg/kg) uponintravenous injection of them. With regard to a reason why theintroduction of a fluorine atom in the 5-position of kanamycin B and itsanalogues can reduce the toxicity of kanamycin B etc., through anymechanism, the present inventors have studied it with groping in thedark. As an outcome, however, the present inventors have now reached ingetting a presumption that the 5-fluoro substitutent can result in areduction in the basicity of the 3-amino group of kanamycins andconsequently result in a reduction in the toxicity of kanamycins.

On the basis of this discovery, the present inventors have now presumedthat, in general, if there can be synthetized such 5-deoxy-5,5-difluoroderivatives of the aminoglycosidic antibiotics in which two fluorosubstituents have been introduced into the 5-position of theaminoglycosidic antibiotics, new semi-synthetic aminoglycosidicantibiotics having a further reduced toxicity can be provided.

DISCLOSURE OF THE INVENTION

The present inventors have made extensive research based on theabove-mentioned presumption, and as a result, have now succeeded insynthetizing as new compounds such 5-deoxy-5,5-difluoro derivatives ofthe aminoglycosidic antibiotics, including those of neamine, kanamycinA-series, kanamycin B-series, gentamicin-series and seldomycin-series.Also, the present inventors have succeeded in synthetizing1-N-(α-hydroxy-ω-aminoalkanoyl) derivatives from5-deoxy-5,5-difluorokanamycins A and B, as well as their analogues.

Further, according to the findings now obtained by the presentinventors, it has been recognized that the 5-deoxy-5,5-difluoroderivatives of the aminoglycosidic antibiotics now synthetized exhibitantibacterial activities enhanced much than or substantially equal tothe antibacterial activities of their respectively corresponding parentaminoglycosidic antibiotics in respect of their minimum growthinhibitory concentrations (MIC.) (as measured according to a standardserial dilution method in vitro) against some species or strains ofbacteria, and that when the aforesaid 5-dideoxy-5,5-difluoro derivativesof the aminoglycosidic antibiotics are intravenously administered tomice as one of mammals, their 50% lethal dosages (LD₅₀) are remarkablyimproved better. Thus, it has been found that the acute toxicity of the5-deoxy-5,5-difluoro derivatives of the aminoglycosidic antibiotics,namely the new compounds now synthetized by the present inventors havebeen reduced to a half or less, as compared to that of the respectivelycorresponding parent aminoglycosidic antibiotics, when they areintravenously administered to mice.

Next, this invention is described in more details. The knownaminoglycosidic antibiotics and their known semi-synthetic derivativesinclude neamine, 3',4'-dideoxyneamine, kanamycin A,3'-deoxykanamycin A,3',4'-dideoxykanamycin A, kanamycin B, 3'-deoxykanamycin B (namely,tobramycin), 3'-deoxy-3'-fluorokanamycin B, 3',4'-dideoxykanamycin B(namely, dibekacin), 3',4'-dideoxy-3'-fluorokanamycin B, gentamicin C₁,gentamicin C_(1a), gentamicin C₂, sagamicin, sisomicin, netilmicin andseldomycin factor 3 etc., which may generically be represented by thegeneral formula ##STR1## wherein R is a hydrogen atom or ethyl group, G¹is either an aminoglycosyl group of the formula ##STR2## where A¹ is ahydroxyl group or amino group, A² and A³ are independently a hydrogenatom, hydroxyl group or fluoro group, A⁴ is a hydrogen atom or methylgroup, and A⁵ is a hydrogen atom or methyl group, or a4'-eno-aminoglycosyl group of the formula ##STR3## where A¹, A², A⁴ andA⁵ independently have the same meanings as defined above, and G² iseither a hydrogen atom, or a 3"-amino-3"-deoxyglycosyl group of theformula ##STR4## where B¹ is a hydrogen atom or methyl group, B² and B³are independently a hydrogen atom, hydroxyl group or methyl group, andB⁴ is a hydrogen atom or a hydroxymethyl group (--CH₂ OH) , or a2"-amino-2"-deoxyglycosyl group of the formula ##STR5## where B¹, B², B³and B⁴ independently have the same meanings as defined above.

To produce synthetically the above-mentioned 5-deoxy-5,5-difluoroderivative of aminoglycosidic antibiotics with starting from4-O-(aminoglycosyl)- or 4,6-di-O-(aminoglycosyl)-2-deoxystreptaminesrepresented by the general formula (A) above, a reaction stage isfirstly conducted, where all the amino groups of the starting2-deoxystreptamine compound of the general formula (A) are protectedaccording to the known amino-protecting methods, such as acylation(e.g., acetylation or benzoylation etc.,), conversion into urethanederivative (e.g., by methoxycarbonylation, ethoxycarbonylation,phenoxycarbonylation or benzyloxycarbonylation etc.,), or sulfonylation(e.g., tosylation etc.,). Subsequently, a reaction stage is conducted,where all the hydroxyl groups other than the 5-hydroxyl group of theamino-protected derivative prepared in the preceeding step are protectedaccording to the known hydroxyl-protecting methods, such as acetylationor benzoylation. At this time, when the starting compound used is suchcompound having 3"-amino group and 4"-hydroxyl group in thecis-configuration, like gentamicins, these amino group and hydroxylgroup can be protected by converting them into the form of a cycliccarbamate by a known procedure. The 5-hydroxyl group of the startingcompound of the general formula (A) can receive the steric hindranceowing to the existence of the aminoglycosyl group at the 4-position orof the two aminoglycosyl groups at the 4- and 6-positions. Therefore,when the starting compound of the formula (A) is subjected to thehydroxyl-protecting reaction, the 5-hydroxyl group is relatively notlikely to be acetylated or benzoylated, so that there can be obtained ina relatively high yield such an N,O-protected 2-deoxystreptaminederivative having the free 5-hydroxyl group and represented by thegeneral formula ##STR6## wherein X is a known amino-protecting groupsuch as an acyl group, an alkoxycarbonyl group, benzyloxycarbonyl groupor a sulfonyl group; G^(1a) denotes an N,O-protected aminoglycosyl groupas derived by protecting all of the amino groups and hydroxyl groups ofthe aminoglycosyl group (G¹) of the formula (i) or of the4'-eno-aminoglycosyl group (G¹) of the formula (ii) shown in theabove-mentioned general formula (A); and G^(2a) denotes an N,O-protectedaminoglycosyl group as derived by protecting all of the amino groups andhydroxyl groups of the 3" -amino-3"-deoxyglycosyl group (G²) of theformula (iii) or of the 2"-amino-2"-deoxyglycosyl group (G²) of theformula (iv) shown in the above-mentioned general formula (A), as willbe referred to repeatedly and similarly hereinafter.

The so prepared compound having the free 5-hydroxyl group andrepresented by the general formula (B) above is then oxidized to producethe corresponding 5-keto derivative of the formula (C) shown below.##STR7##

When the above-mentioned oxidation is effected by reacting the compoundof the formula (B) with a known oxidizing agent such as pyridiniumchlorochromate (PCC) or a mixture of dimethylsulfoxide (DMSO) and aceticanhydride, the 5-hydroxyl group of the compound (B) is oxidized toproduce the 5-keto derivative of the formula (C). This oxidationreaction may suitably be conducted by using dichloromethane,dimethylsulfoxide (DMSO), pyridine, a mixture of pyridine-DMSO, benzene,carbon tetrachloride, chloroform, acetonitrile and the like as thesolvent. The reaction temperature for this oxidation may suitably be ina range of -20° C. to 100° C. The oxidation reaction can be finished inone week. The 5-keto derivative of the formula (C) is then reacted witha fluorinating agent such as DAST as a typical example, in an organicsolvent such as dichloromethane, benzene, dichloromethane-pyridine,carbon tetrachloride, chloroform, acetonitrile and the like at atemperature of 0° to 100° C., with the fluorinating agent being used ina proportion of 5 to 20 moles per mole of the 5-keto derivative.Thereby, the 2,5-dideoxy-5,5-difluorostreptamine derivative of theformula ##STR8## wherein X, G^(1a) and G^(2a) have the same meanings asdefined above is produced and obtained in a relatively high yield.Although it is generally known that DAST is reacted with a keto grouppresent in an organic compound to introduce difluoro groups into theorganic compound, it is achieved firstly up to dates by the presentinventors that a keto derivative is prepared from the aminoglycosidicantibiotics and then reacted with DAST with getting a success insynthetizing the 5-deoxy-5,5-difluoro derivatives of theaminoglycosides.

From the 2,5-dideoxy-5,5-difluorostreptamine derivative of the formula(D) are then removed the amino-protecting groups and thehydroxyl-protecting groups by known methods (for example, byde-acylation with alkaline treatment, de-alkoxycarbonylation,de-benzyloxycarbonylation through catalytic reduction, orde-N-tosylation by treatment with sodium in liquefied ammonia, etc.),whereby there can be obtained the desired2,5-dideoxy-5,5-difluorostreptmine derivative of the formula ##STR9##wherein R, G¹ and G² have the same meanings as defined in the abovegeneral formula (A). From amongst the 4-O-(aminoglycosyl)- or4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamines of theformula (I¹) above, is taken such a compound of the formula ##STR10##wherein G¹ and G² have the same meanings as defined in the above generalformula (A), as a compound having unsubstituted 1-amino group, namelysuch compound where R is a hydrogen atom. The compound of the formula(I²) is then treated by the selectively amino-protecting methoddisclosed in the specification of U.S. Pat. No. 4,297,485, so that allor some of the amino groups of the compound of the formula (I²) areprotected with appropriate amino-protecting groups, and thereby it isfeasible to prepare a1-N-unprotected-poly-N-protected-4-O-(aminoglycosyl)- or4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamine representedby the formula ##STR11## wherein X' is appropriate amino-protectinggroup; and G^(1b) denotes an N-protected aminoglycosyl group as derivedby protecting with the amino-protecting group (X') all or some of theamino groups of the aminoglycosyl group (G¹) of the formula (i) or ofthe 4'-eno-aminoglycosyl group (G¹) of the formula (ii) shown in theabove-mentioned general formula (A); and G^(2b) denotes an N-protectedaminoglycosyl group as derived by protecting with appropriateamino-protecting group all or some of the amino groups of the3"-amino-3"-deoxyglycosyl group (G²) of the formmla (iii) or of the2"-amino-2"-deoxyglycosyl group (G²) of the formula (iv) shown in theabove-mentioned general formula (A).

When the 1-amino group of the compound of the formula (I³) is eitheralkylated by reacting with an alkyl bromide or iodide of the formula

    R.sup.1 --Hal                                              (X)

where R¹ is an alkyl group of 1 to 4 carbon atoms and Hal denotesbromine or iodine, or acylated by reaction with anα-hydroxy-ω-aminoalkanoic acid of the formula ##STR12## where n is aninteger of 1 to 3, for example, 3-amino-2-(RS)-hydroxypropionic acid or4-amino-2-(S)-hydroxybutyric acid, or with its protected derivativehaving the amino group protected with an amino-protective group or itsactivated derivative, there can be produced a 1-N-modified derivativehaving the formula ##STR13## wherein X', G^(1b) and G^(2b) have the samemeanings as defined above and R¹ is an alkyl group of 1 to 4 carbonatoms or an α-hydroxy-ω-aminoalkanoyl group of the formula ##STR14##where n is an integer of 1 to 3.

By removing the amino-protecting groups from the compound of the formula(I⁴) by conventional methods, a 1-N-alkyl- or1-N-(α-hydroxy-ω-aminoalkanoyl)-4-O-(aminoglycosyl)- or-4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamine having theformula ##STR15## wherein R¹, G¹ and G² have the same meanings asdefined above can be produced.

The compound represented by the formula (I¹) above, as well as thecompound represented by the formula (I⁵) are included by the new5-deoxy-5,5-difluoro derivatives of the aminoglycosidic antibiotics asdesired and provided in accordance with this invention.

According to the first aspect of this invention, therefore, there isprovided a 4-O-(aminoglycosyl)- or4,6di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamine derivativerepresented by the general formula ##STR16## wherein R¹ is a hydrogenatom, an alkyl group of 1-4 carbon atoms or an α-hydroxy-ω-aminoalkanoylgroup of the formula ##STR17## where n is an integer of 1 to 3, G¹ iseither an aminoglycosyl group of the formula ##STR18## Where A¹ is ahydroxyl group or amino group, A² and A³ are independently a hydrogenatom, hydroxyl group or fluoro group, A⁴ is a hydrogen atom or methylgroup, and A⁵ is a hydrogen atom or methyl group, or a4'-eno-aminoglycosyl group of the formula ##STR19## where A¹, A², A⁴ andA⁵ independently have the same meanings as defined above, and G² iseither a hydrogen atom, or a 3"-amino-3"-deoxyglycosyl group of theformula ##STR20## where B¹ is a hydrogen atom or methyl group, B² and B³are independently a hydrogen atom, hydroxyl group or methyl group, andB⁴ is a hydrogen atom or a hydroxymethyl group (--CH₂ OH), or a2"-amino-2"-deoxyglycosyl group of the formula ##STR21## where B¹, B²,B³ and B⁴ independently have the same meanings as defined above, and apharmaceutically acceptable acid addition salt of said derivative.

The new compound of the general formula (I) according to this inventionis usually obtained in the form of a free base, a hydrate or a carbonatethereof. The new compound of the formula (I) may, if desired, beconverted into a pharmaceutically acceptable acid addition salt thereofin a known manner. Such acid addition salts of said compound includesuch salt with a pharmaceutically acceptable inorganic acid such ashydrochloric acid, sulfuric acid, phosphoric acid, nitric acid and thelike; or with a pharmaceutically acceptable organic acid such as aceticacid, malic acid, citric acid, ascorbic acid, methanesulfonic acid andthe like.

A compound of the general formula (I) according to this invention or anacid addition salt thereof may be mixed with a pharmaceuticallyacceptable liquid or solid carrier or vehicle to prepare anantibacterial composition.

The 2,5-dideoxy-5,5-difluorostreptamine derivative of the generalformula (I) according to this invention may include a streptaminederivative which is a4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamine derivativerepresented by the general formula ##STR22## wherein A¹, A², A³, A⁴ andA⁵, as well as B¹, B², B³ and B⁴ have the same meanings as defined forthe general formula (I) described above, and which is such a compoundbelonging to a derivative of kanamycin A, a derivative of kanamycin B, aderivative of gentamicin C₁, a derivative of gentamicin C_(1a) or aderivative of gentamicin C₂ or a derivative of sagamicin.

The streptamine derivative of the general formula (Ia) above may largelybe classified as its preferred embodiments into the compounds of threeclasses which are respectively represented by the following formula(Ia-1), formula (Ia-2) and formula (Ia-3):

(1) A streptamine derivative which is a compound represented by thegeneral formula ##STR23## wherein (i) A² and A³ are each a hydroxylgroup, or (ii) A² is a hydrogen atom and A³ is a hydroxyl group, or(iii) A² and A³ are each a hydrogen atom, and which is namely (i)5-deoxy-5,5-difluorokanamycin A, or (ii)5,3'-dideoxy-5,5-difluorokanamycin A, or (iii)5,3',4'-trideoxy-5,5-difluorokanamycin A.

(2) A streptamine derivative which is a compound represented by thegeneral formula ##STR24## wherein (i) A² and A³ are each a hydroxylgroup, or (ii) A² is a hydrogen atom and A³ is a hydroxyl group, or(iii) A² and A³ are each a hydrogen atom, or (iv) A² is a fluoro groupand A³ is a hydroxyl group, or (v) A² is a fluoro group and A³ is ahydrogen atom, and which is namely (i) -deoxy-5,5-difluorokanamycin B,or (ii) 5,3'-dideoxy-5,5 -difluorokanamycin B, or (iii)5,3',4'-trideoxy-5,5-difluorokanamycin B, or (iv)5,3'-dideoxy-5,5,3'-trifluorokanamycin B, or (v)5,3',4'-trideoxy-5,5,3'-trifluorokanamycin B.

(3) A streptamine derivative which is a compound represented by thegeneral formula ##STR25## wherein (i) A² and A³ are each a hydrogen atomand A⁴ and A⁵ are each a methyl group, or (ii) A², A³, A⁴ and A⁵ are alland each a hydrogen atom, or (iii) A², A³ and A⁵ are all and each ahydrogen atom and A⁴ is methyl group, or (iv) A², A³ and A⁴ are all andeach a hydrogen atom and A⁵ is methyl group, and which is namely (i)5-deoxy-5,5-difluorogentamicin C₁, or (ii)5-deoxy-5,5-difluorogentamicin C_(1a), or (iii)5-deoxy-5,5-difluorogentamicin C₂, or (iv)5-deoxy-5,5-difluorosagamicin.

Further, the 2,5-dideoxy-5,5-difluorostreptamine derivative of thegeneral formula (I) according to this invention may include astreptamine derivative which is a compound represented by the generalformula ##STR26## wherein (i) R¹ is a hydrogen atom and A², A⁴ and A⁵are all and each a hydrogen atom, or (ii) R¹ is ethyl group and A², A⁴and A⁵ are all and each a hydrogen atom, and which is namely (i)5-deoxy-5,5-difluorosisomicin or (ii) 5-deoxy-5,5-difluoronetilmicin.

Furthermore, the 2,5-dideoxy-5,5-difluorostreptamine derivative of thegeneral formula (I) according to this invention may include astreptamine derivative which is a1-N-(α-hydroxy-ω-aminoalkanoyl)-4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptaminederivative represented by the general formula ##STR27## wherein n is aninteger of 1 to 3, and A¹, A², A³, A⁴ and A⁵ as well as B¹, B², B³ andB⁴ respectively have the same meanings as defined for the generalformula (I) described above or for the general formula (Ia) describedabove.

The streptamine derivative of the general formula (Ic) above may largelybe classified as its preferred embodiments into the compounds of threeclasses which are respectively represented by the following formula(Ic-1), formula (Ic-2) and formula (Ic-3):

(1) A streptamine derivative which is a compound represented by thegeneral formula ##STR28## wherein n is an integer of 1 to 3, and (i) A²and A³ are each a hydroxyl group, or (ii) A² is a hydrogen atom and A³is hydroxyl group, or (iii) A² and A³ are each a hydrogen atom, andwhich is namely a 1-N-(α-hydroxy-ω-aminoalkanoyl) derivative of anyoneof (i) 5-deoxy-5,5-difluorokanamycin A or (ii)5,3'-dideoxy-5,5-difluorokanamycin A or (iii)5,3',4'-trideoxy-5,5-difluorokanamycin A.

(2) A streptamine derivative which is a compound represented by thegeneral formula ##STR29## wherein n is an integer of 1 to 3, and (i) A²and A³ are each a hydroxyl group, or (ii) A² is a hydrogen atom and A³is hydroxyl group, or (iii) A² and A³ are each a hydrogen atom, or (iv)A² is a fluoro group and A³ is hydroxyl group, or (v) A² is a fluorogroup and A³ is a hydrogen atom, and which is namely a1-N-(α-hydroxy-ω-aminoalkanoyl) derivative of anyone of (i)5-deoxy-5,5-difluorokanamycin B or (ii)5,3'-dideoxy-5,5-difluorokanamycin B or (iii)5,3',4'-trideoxy-5,5-difluorokanamycin B or (iv)5,3'-dideoxy-5,5,3'-trifluorokanamycin B or (v)5,3',4'-trideoxy-5,5,3'-trifluorokanamycin B.

(3) A streptamine derivative which is a compound represented by thegeneral formula ##STR30## wherein n is an integer of 1 to 3, and (i) A²and A³ are each a hydrogen atom and A⁴ and A⁵ are each a methyl group,or (ii) A², A³, A⁴ and A⁵ are all and each a hydrogen atom, or (iii) A²,A³ and A⁵ are all and each a hydrogen atom and A⁴ is methyl group, or(iv) A², A³ and A⁴ are all and each a hydrogen atom and A⁵ is methylgroup, and which is a 1-N-(α-hydroxy-ω-aminoalkanoyl) derivative ofanyone of (i) 5-deoxy-5,5-difluorogentamicin C₁ or (ii)5-deoxy-5,5-difluorogentamicin C_(1a) or (iii)5-deoxy-5,5-difluorogentamicin C₂ or (iv) 5-deoxy-5,5-difluorosagamicin.

Moreover, the 2,5-dideoxy-5,5-difluorostreptamine derivative of thegeneral formula (I) according to this invention may include astreptamine derivative which is 5-deoxy-5,5-difluoroseldomycin factor 3represented by the formula ##STR31##

Next, according to the second aspect of this invention, there isprovided an antibacterial composition, characterized in that saidcomposition comprises a 4-O-(aminoglycosyl)- or4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamine derivativerepresented by the general formula ##STR32## wherein R¹, G¹ and G²respectively have the same meanings as defined for the general formula(I) shown hereinbefore, as the active ingredient, in association with apharmaceutically acceptable carrier for the active ingredient.

The antibacterial activity of the compounds of the general formula (I)according to this invention has been examined by determining theirminimum inhibitory concentrations (MIC, mcg/ml) against the growth ofvarious kinds of bacteria by a standard serial dilution method (onMueller-Hinton agar medium incubated at 37° C).

Among the compounds of the general formula (I) according to thisinvention, such representative compounds of which the MIC. has beendetermined are listed below. All of these compounds are in the form ofbasic and colorless and powdery substance having no definite meltingpoint.

(1) 5-deoxy-5,5,-difluorokanamycin A (abbreviated as 5FF-KMA).

(2) 1-N-[4-amino-2-(S)-hydroxybutyryl]-5-deoxy-5,5-difluorokanamycin A,namely 5-deoxy-5,5-difluoroamikacin (abbreviated as 5FF-amikacin).

(3) 5-deoxy-5,5-difluorokanamycin B (abbreviated as 5FF-KMB).

(4) 5,3'-dideoxy-5,5-difluorokanamycin B, namely5-deoxy-5,5-difluorotobramycin (abbreviated as 5FF-TOB).

(5) 1-N-[4-amino-2-(S)-hydroxybutyryl]-5-deoxy-5,5-difluorotobramycin(abbreviated as 5FF-TOB-AHB).

(6) 5,3',4'-trideoxy-5,5-difluorokanamycin B, namely5-deoxy-5,5-difluorodibekacin (abbreviated as 5FF-DKB).

(7)1-N-[4-amino-2-(S)-hydroxybutyryl]-5,3',4'-trideoxy-5,5-difluorokanamycinB (abbreviated as 5FF-DKB-AHB).

(8) 5-deoxy-5,5-difluorogentamicin C₁ (abbreviated as 5FF-genta-C₁).

(9) 1-N-[4-amino-2-(S)-hydroxybutyryl]-5-deoxy-5,5-difluorogentamicin C₁(abbreviated as 5FF-genta-C₁ -AHB).

(10) 5-deoxy-5,5-difluorogentamicin C₂ (abbreviated as 5FF-genta-C₂).

(11) 1-N-[4-amino-2-(S)-hydroxybutyryl]-5-deoxy-5,5-difluorogentamicinC₂ (abbreviated as 5FF-genta-C₂ -AHB).

(12) 5-deoxy-5,5-difluorogentamicin C_(1a) (abbreviated as5FF-genta-C_(1a)).

(13) 5-deoxy-5,5-difluoronetilmicin (abbreviated as 5FF-netilmicin).

The antibacterial spectra (MIC values, mcg/ml) so determined of theabove-mentioned respective compounds are shown in Table 1-a to Table 1-cbelow. For comparison, the antibacterial spectra (MIC values, mcg/ml)similarly determined of kanamycin A (abbreviated as KMA), kanamycin B(abbreviated as KMB), amikacin, 3'-deoxykanamycin B, namely tobramycin(abbreviated as TOB), 5,3'-dideoxy-5-fluorokanamycin B (abbreviated as5F-TOB),1-N-[4-amino-2-(S)-hydroxybutyryl]-5,3'-dideoxy-5-fluorokanamycin B(abbreviated as 5F-TOB-AHB), 3',4'-dideoxy-kanamycin B (abbreviated asDKB), 5,3',4'-trideoxy-5-fluorokanamycin B (abbreviated as 5F-DKB),1-N-[4-amino-2-(S)-hydroxybutyryl]-5,3',4'-trideoxy-5-fluorokanamycin B(abbreviated as 5F-DKB-AHB), gentamicin C₁ (abbreviated as genta-C₁),gentamicin C₂ (abbreviated as genta-C₂) and gentamicin C_(1a)(abbreviated as genta-C_(1a)) are also shown in Table 1-a to Table 1-cbelow.

                                      TABLE 1-a                                   __________________________________________________________________________                                 MIC. (mcg/ml)                                                      Resistance KMA     5FF-KMA                                  Tested Microorganisms                                                                           mechanism  (Comparative)                                                                         (Invention)                              __________________________________________________________________________    Staphylococcus aureus 209P   1.56    3.12                                     Staphylococcus aureus Ap01                                                                      ANT(4')    25      50                                       Bacillus subtilis PCI219     0.78    0.78                                     Corynebacterium bovis 1810   12.5    12.5                                     Escherichia coli K-12        0.78    0.78                                     Escherichia coli K-12 R5                                                                        AAC(6')    >100    >100                                     Escherichia coli K-12 ML 1629                                                                   APH(3')-I  >100    >100                                     Escherichia coli K-12 LA 290 R55                                                                ANT(2")    50      12.5                                     Escherichia coli JR225                                                                          AAC(3)     3.12    1.56                                                        APH(3')-II                                                 Escherichia coli JR66/W677   >100                                                               ANT(2")                                                     Mycobacterium 607            6.25    3.12                                                        APH(3')-II                                                 Klebsiella pneumoniae 22#3038                                                                              >100                                                               ANT(2")                                                     Proteus rettgeri GN 311      0.78    1.56                                     Serratia marcescens          12.5    12.5                                     Providencia sp. Pv 16                                                                           AAC(2')    3.12    12.5                                     Pseudomonas aeruginosa A3    6.25    6.25                                     Pseudomonas aeruginosa GN 315                                                                   AAC(6')    >100    >100                                     __________________________________________________________________________                            MIC. (mcg/ml)                                                           Resistance                                                                          Amikacin                                                                              5FF-amikacin                                                                         KMB     5FF-KMB                        Tested Microorganisms                                                                           mechanism                                                                           (Comparative)                                                                         (invention)                                                                          (Comparative)                                                                         (Invention)                    __________________________________________________________________________    Staphylococcus aureus 209P                                                                            0.78    3.12   0.39    0.78                           Staphylococcus aureus Ap01                                                                      ANT(4')                                                                             >100    >100   25      50                             Bacillus subtilis PCI219                                                                              0.39    1.56   <0.2    0.39                           Corynebacterium bovis 1810                                                                            1.56    3.12   0.78    1.56                           Escherichia coli K-12   0.78    1.56   0.39    0.39                           Escherichia coli K-12 R5                                                                        AAC(6')                                                                             100     25     50      100                            Escherichia coli K-12 ML 1629                                                                   APH(3')-I                                                                           1.56    3.12   >100    >100                           Escherichia coli K-12 LA 290 R55                                                                ANT(2")                                                                             1.56    3.12   12.5    3.12                           Escherichia coli JR225                                                                          AAC(3)                                                                              0.78    0.39   12.5    3.12                                              APH(3')-II                                                 Escherichia coli JR66/W677                                                                            3.12    3.12   >100                                                     ANT(2")                                                     Mycobacterium 607       0.78    3.12   1.56    1.56                                               APH(3')-II                                                Klebsiella pneumoniae 22#3038                                                                         3.12    6.25   >100                                                     ANT(2")                                                     Proteus rettgeri GN 311 1.56    1.56   0.2     0.39                           Serratia marcescens     3.12    6.25   3.12    6.25                           Providencia sp. Pv 16                                                                           AAC(2')                                                                             1.56    3.12   12.5    12.5                           Pseudomonas aeruginosa A3                                                                             0.78    3.12   1.56    3.12                           Pseudomonas aeruginosa GN 315                                                                   AAC(6')                                                                             50      >100   50      >100                           __________________________________________________________________________

                                      TABLE 1-b                                   __________________________________________________________________________                            MIC. (mcg/ml)                                                           Resistance                                                                          TOB     5F-TOB  5FF-TOB                                                                             5F-TOB-AHB                                                                            5FF-TOB-AHB             Tested Microorganisms                                                                           mechanism                                                                           (Comparative)                                                                         (Comparative)                                                                         (Invention)                                                                         (Comparative)                                                                         (Invention)             __________________________________________________________________________    Staphylococcus aureus 209P                                                                            <0.2    0.39    0.39  0.39    0.39                    Staphylococcus aureus Ap01                                                                      ANT(4')                                                                             12.5    0.39    25    3.12    3.12                    Bacillus subtilis PCI219                                                                              <0.2    <0.2    0.39  <0.2    <0.2                    Corynebacterium bovis 1810                                                                            1.56    1.56    1.56  <0.2    <0.2                    Escherichia coli K-12   0.39    <0.2    0.39  <0.2    <0.2                    Escherichia coli K-12 R5                                                                        AAC(6')                                                                             25      25      25    3.12    3.12                    Escherichia coli K-12 ML 1629                                                                   APH(3')-I                                                                           0.78    0.78    0.78  0.39    0.39                    Escherichia coli K-12 LA 290 R55                                                                ANT(2")                                                                             12.5    3.12    0.78  0.39    <0.2                    Escherichia coli JR225                                                                          AAC(3)                                                                              25      0.78    0.39  <0.2    <0.2                                       APH(3')-II                                                 Escherichia coli JR66/W677                                                                            12.5    0.78    1.56  0.78    0.39                                      ANT(2")                                                     Mycobacterium 607       0.39    0.78    0.39  0.39    0.39                                       APH(3')-II                                                 Klebsiella pneumoniae 22#3038                                                                         12.5    3.12    0.78  0.78    0.78                                      ANT(2")                                                     Proteus rettgeri GN 311 0.39    <0.2    <0.2  <0.2    0.39                    Serratia marcescens     3.12    6.25    3.12  3.12    1.56                    Providencia sp. Pv 16                                                                           AAC(2')                                                                             6.25    3.12    12.5  0.39    0.78                    Pseudomonas aeruginosa A3                                                                             <0.2    <0.2    <0.2  <0.2    0.78                    Pseudomonas aeruginosa GN 315                                                                   AAC(6')                                                                             25      50      100   1.56    6.25                    __________________________________________________________________________                            MIC. (mcg/ml)                                                           Resistance                                                                          DKB     5F-DKB  5FF-DKB                                                                             5F-DKB-AHB                                                                            5FF-DKB-AHB             Tested Microorganisms                                                                           mechanism                                                                           (Comparative)                                                                         (Comparative)                                                                         (Invention)                                                                         (Comparative)                                                                         (Invention)             __________________________________________________________________________    Staphylococcus aureus 209P                                                                            0.39    0.78    0.78  0.39    0.39                    Staphylococcus aureus Ap01                                                                      ANT(4')                                                                             0.78    3.12    3.12  1.56    3.12                    Bacillus subtilis PCI219                                                                              <0.2    0.78    0.39  <0.2    0.39                    Corynebacterium bovis 1810                                                                            6.25    6.25    1.56  <0.2    <0.2                    Escherichia coli K-12   0.39    3.12    0.39  <0.2    <0.2                    Escherichia coli K-12 R5                                                                        AAC(6')                                                                             50      100     100   12.5    3.12                    Escherichia coli K-12 ML 1629                                                                   APH(3')-I                                                                           0.78    3.12    0.78  0.39    0.39                    Escherichia coli K-12 LA 290 R55                                                                ANT(2")                                                                             50      6.25    1.56  0.39    <0.2                    Escherichia coli JR225                                                                          AAC(3)                                                                              100     3.12    3.12  <0.2    <0.2                                       APH(3')-II                                                 Escherichia coli JR66/W677                                                                            25      3.12    1.56  0.78    0.39                                      ANT(2")                                                     Mycobacterium 607       1.56    6.25    1.56  0.78    0.78                                       APH(3')-II                                                 Klebsiella pneumoniae 22#3038                                                                         50      6.25    1.56  0.78    0.78                                      ANT(2")                                                     Proteus rettgeri GN 311 <0.2    0.78    0.39  <0.2    0.39                    Serratia marcescens     25      12.5    12.5  3.12    1.56                    Providencia sp. Pv 16                                                                           AAC(2')                                                                             25      12.5    12.5  0.39    0.78                    Pseudomonas aeruginosa A3                                                                             0.2     0.39    0.39  0.39    0.78                    Pseudomonas aeruginosa GN 315                                                                   AAC(6')                                                                             100     100     >100  6.25    12.5                    __________________________________________________________________________

                                      TABLE 1-c                                   __________________________________________________________________________                            MIC. (mcg/ml)                                                                                 5FF-Genta-                                              Resistance                                                                          Genta-C.sub.1                                                                         5FF-Genta-C.sub.1                                                                     C.sub.1 -AHB                                                                        Genta-C.sub.2                                                                         5FF-Genta-C.sub.2       Tested Microorganisms                                                                           mechanism                                                                           (Comparative)                                                                         (Invention)                                                                           (Invention)                                                                         (Comparative)                                                                         (Invention)             __________________________________________________________________________    Staphylococcus aureus 209P                                                                            <0.2    1.56    1.56  <0.2    0.78                    Staphylococcus aureus Ap01                                                                      ANT(4')                                                                             0.78    12.5    25    0.78    6.25                    Bacillus subtilis PCI219                                                                              <0.2    1.56    1.56  <0.2    0.2                     Corynebacterium bovis 1810                                                                            0.39    3.12    0.78  0.39    0.78                    Escherichia coli K-12   0.2     1.56    <0.2  <0.2    0.78                    Escherichia coli K-12 R5                                                                        AAC(6')                                                                             0.78    1.56    0.78  1.56    1.56                    Escherichia coli K-12 ML 1629                                                                   APH(3')-I                                                                           0.39    1.56    1.56  0.39    0.78                    Escherichia coli K-12 LA 290 R55                                                                ANT(2")                                                                             12.5    0.78    0.39  12.5    3.12                    Escherichia coli JR225                                                                          AAC(3)                                                                              25      12.5    <0.2  6.25    6.25                                       APH(3')-II                                                 Escherichia coli JR66/W677                                                                            12.5    0.78    3.12  25      3.12                                      ANT(2" )                                                    Mycobacterium 607       3.12    1.56    3.12  1.56    0.78                                       APH(3')-II                                                 Klebsiella pneumoniae 22#3038                                                                         12.5    3.12    1.56  12.5    1.56                                      ANT(2")                                                     Porteus rettgeri GN 311 0.78    1.56    1.56  0.2     0.78                    Serratia marcescens     0.78    1.56    3.12  0.78    0.78                    Providencia sp. Pv 16                                                                           AAC(2')                                                                             25      25      6.25  6.25    6.25                    Pseudomonas aeruginosa A3                                                                             0.78    6.25    6.25  0.39    1.56                    Pseudomonas aeruginosa GN 315                                                                   AAC(6')                                                                             6.25    25      100   3.12    25                      __________________________________________________________________________                            MIC. (mcg/ml)                                                                 5FF-Genta-      5FF-Genta-                                              Resistance                                                                          C.sub.2 -AHB                                                                          Genta-C.sub.1a                                                                        C.sub.1a                                                                            5FF-Netilmicin                                                                        Netilmicin              Tested Microorganisms                                                                           mechanism                                                                           (Invention)                                                                           (Comparative)                                                                         (Invention)                                                                         (Invention)                                                                           (Comparative)           __________________________________________________________________________    Staphylococcus aureus 209P                                                                            0.78    <0.2    0.78  3.12    <0.2                    Staphylococcus aureus Ap01                                                                      ANT(4')                                                                             12.5    0.39    3.12  12.5    0.78                    Bacillus subtilis PCI219                                                                              0.2     <0.2    0.39  0.39    <0.2                    Corynebacterium bovis 1810                                                                            0.2     <0.2    0.78  1.56    1.56                    Escherichia coli K-12   <0.2    <0.2    <0.2  <0.2    <0.2                    Escherichia coli K-12 R5                                                                        AAC(6')                                                                             3.12    3.12    12.5  50      50                      Escherichia coli K-12 ML 1629                                                                   APH(3')-I                                                                           0.39    0.39    0.39  0.78    0.39                    Escherichia coli K-12 LA 290 R55                                                                ANT(2")                                                                             0.2     >100    3.12  0.39    0.39                    Escherichia coli JR225                                                                          AAC(3)                                                                              <0.2    25      3.12  1.56    12.5                                       APH(3')-II                                                 Escherichia coli JR66/W677                                                                            0.78    12.5    1.56  0.78    0.39                                      ANT(2")                                                     Mycobacterium 607       3.12    6.25    0.78  1.56    1.56                                       APH(3')-II                                                 Klebsiella pneumoniae 22#3038                                                                         1.56    12.5    1.56  1.56    0.39                                      ANT(2")                                                     Proteus rettgeri GN 311 0.78    <0.2    0.39  1.56    0.39                    Serratia marcescens     3.12    1.56    6.25  6.25    6.25                    Providencia sp. Pv 16                                                                           AAC(2')                                                                             6.25    12.5    12.5  12.5    6.25                    Pseudomonas aeruginosa A3                                                                             3.12    <0.2    0.78  1.56    0.39                    Pseudomonas aeruginosa GN 315                                                                   AAC(6')                                                                             100     12.5    100   >100    >100                    __________________________________________________________________________

As will be clear from the antibacterial data of Table 1, the compound ofthe general formula (I) according to this invention can exhibit its highantibacterial activity against many species of bacteria and has broadantibacterial spectra.

Further, in order to estimate acute toxicity of the compound of thegeneral formula (I) according to this invention, some examples of thecompounds of this invention were independently dissolved in water toprepare aqueous solutions (as adjusted to pH 7 by addition ofhydrochloric acid). Each of these aqueous solutions was intravenouslyinjected to mice of dd-strain (male, 4 week-aged, four mice per group)and then 50% lethal dosage (LD₅₀) (mg/kg) of the tested compound wasevaluated. The tested compounds of this invention are expressed by thesame abbreviations as in Table 1. The acute toxicity of some examples ofthe known, comparative compounds was evaluated by a similar tests. Thecomparative compounds as tested are also expressed by the sameabbreviations as in Table 1. While, "HBK" denotes1-N-[4-amino-(S)-2-hydroxybutyryl]-3',4'-dideoxykanamycin B.5FF-netilmicin denotes 5-deoxy-5,5-difluoronetilmicin according to thisinvention.

The results of the above tests for estimation of the acute toxicity ofthe compounds are summarized in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Tested compounds      LD.sub.50 (mg/kg)                                       ______________________________________                                        5FF-KMA       (this Invention)                                                                          >300                                                5FF-amikacin  (this Invention)                                                                          >300                                                Amikacin      (comparative)                                                                             >300                                                5FF-KMB       (this Invention)                                                                          --                                                  DKB           (comparative)                                                                             ca.90                                               5F-DKB        (comparative)                                                                             ca.135                                              HBK           (comparative)                                                                             70                                                  5FF-DKB       (this Invention)                                                                          250                                                 5FF-DKB-AHB   (this Invention)                                                                          ca.200                                              TOB           (comparative)                                                                             ca.80                                               5F-TOB        (comparative)                                                                             ca.180                                              5FF-TOB       (this Invention)                                                                          260                                                 5FF-TOB-AHB   (this Invention)                                                                          >300                                                Genta-C.sub.1 (comparative)                                                                             ca.70                                               5FF-Genta-C.sub.1                                                                           (this Invention)                                                                          ca.200                                              Genta-C.sub.2 (comparative)                                                                             ca.90                                               5FF-genta-C.sub.2                                                                           (this Invention)                                                                          ca.230                                              Genta-C.sub.1a                                                                              (comparative)                                                                             ca.60                                               5FF-genta-C.sub.1a                                                                          (this Invention)                                                                          ca.240                                              Netilmicin    (comparative)                                                                             30                                                  5FF-netilmicin                                                                              (this Invention)                                                                          47                                                  ______________________________________                                    

BEST EMBODIMENTS FOR WORKING THE INVENTION

For the preparation of the compound having the general formula (Ia)amongst the compounds of the general formula (I) according to thisinvention, there is provided according to the third aspect of thisinvention a process for the production of a4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamine derivativerepresented by the general formula ##STR33## wherein A¹, A², A³, A⁴ andA⁵ as well as B¹, B², B³ and B⁴ respectively have the same meanings asdefined for the formula (Ia) described as above, characterized in thatthe process comprises reacting an N,O-protected 5-keto derivative ofkanamycin A, a deoxykanamycin A, a dideoxykanamycin A, kanamycin B, adeoxykanamycin B, a dideoxykanamycin B, 3'-fluoro-3'-deoxykanamycin B,3' -fluoro-3', 4'-dideoxykanamycin B, gentamicin C₁, gentamicin C_(1a)or gentamicin C₂ or sagamicin represented by the general formula##STR34## wherein X and X' respectively are amino-protecting groupswhich may be the same or different; Y is a hydroxyl-protecting group;A^(a) is a protected hydroxyl group or a protected amino group; A^(b)and A^(c) respectively are a hydrogen atom, a protected hydroxyl groupor a fluoro group; A^(d) is a hydrogen atom or methyl group, namely issame as A⁴ set forth in the general formula (I) above; A^(e) is ahydrogen,atom or methyl group, namely- is same as A⁵ set forth in thegeneral formula (I); and B^(a) is a hydrogen atom or methyl group,namely is same as B¹ set forth in the general formula (I); B^(b) andB^(c) respectively are a hydrogen atom, a protected hydroxyl group (OY)or a protected hydroxyl group (OY') as protected by a differenthydroxyl-protecting group, or a methyl group; B^(d) is a hydrogen atomor a hydroxymethyl group as protected at its hydroxyl moiety and havingformula (--CH₂ OY); and Y' is a hydroxyl-protecting group different fromthe group Y, with a dialkylaminosulfur trifluoride of the formula##STR35## wherein R² is an alkyl group of 1 to 4 carbon atoms, or with abis(dialkylamino)sulfur difluoride of the formula

    R.sup.2 --N--SF.sub.2 --NR.sup.2                           (III')

wherein R² is as defined above, or with a fluorination agent equivalentto the compound of formula (III) or (III') in a non-polar organicsolvent, to di-fluorinate the ketone group at the 5-position of the5-keto derivative of the formula (II), thereby producing anN,O-protected 2,5-dideoxy-5,5-difluorostreptamine derivative representedby the general formula ##STR36## wherein X, X', Y, A^(a), A^(b), A^(c),A^(d), A^(e), B^(a), B^(b), B^(c) and B^(d) respectively have the samemeanings as defined above, and then removing the remainingamino-protecting groups (X, X') and the remaining hydroxyl-protectinggroups (Y, Y') from the compound of the formula (IV) by a conventionalmethod.

The method for preparation of the N,O-protected 5-keto derivative of theformula (II) which is used as a starting compound in the process of thethird aspect of this invention is briefly described hereinafter in thisspecification.

In the starting compound of the formula (II) employed in theabove-mentioned process of this invention, all the amino groups born bysaid compound have been protected by any known and appropriateamino-protecting groups (X, X') which do not participate in thereactions involved and which may be, for example, an acyl group,including an alkanoyl group such as acetyl and trifluoroacetyl; and anaroyl group such as benzoyl; an alkoxycarbonyl group such asmethoxycarbonyl, ethoxycarbonyl and butoxycarbonyl; anaralkyloxycarbonyl group such as benzyloxycarbonyl andphenetyloxycarbonyl; or an aryloxycarbonyl group such as phenoxycarbonyland methoxyphenoxycarbonyl; and a protective group of sulfonyl-type,including e.g. an alkylsulfonyl group, an aralkylsulfonyl group such asbenzylsulfonyl and an arylsulfonyl group such as tosyl.

In cases where the starting compound of formula (II) has the hydroxylgroups (A^(a), A^(b) and/or A^(c)) e.g. at the 2', 3'- and/or4'-position thereof in addition to the 5-hydroxyl group, it is necessarythat for instance, all of the 2'-hydroxyl group, 3'-hydroxyl groupand/or 4'-hydroxyl group and the 2"-hydroxyl group other than the5-hydroxyl group should be protected by a known hydroxyl-protectinggroups (Y, Y') which may be selected from an acyl group, preferably analkanoyl or an aroyl group. Similarly, where the 4"- and 6"-hydroxylgroups are present, they are necessary to have been protected by knownhydroxyl-protecting groups (Y, Y') which may be selected from an acylgroup, preferably an alkanoyl or an aroyl group. The acyl group for theprotection of the hydroxyl groups may be an alkanoyl group, typically analkanoyl group of 2-5 carbon atoms such as acetyl, propionyl andbutyryl. Acetyl group is preferred. The acyl group may be an aroylgroup, which includes phenylcarbonyl group optionally having alkylsubstituent(s) on the phenyl ring, preferably benzoyl. Alternatively,the 4"-hydroxyl-protecting group (Y) and 6"-hydroxyl-protecting group(Y) as taken together may form a single known di-valenthydroxyl-protecting group, for example, an alkylidene group of 2-8carbon atoms such as ethylidene and isopropylidene, or a cycloalkylidenegroup such as cyclohexylidene, or a tetrahydropyranylidene group.

Examples of the dialkylaminosulfur trifluoride of formula (III), whichmay be used as the fluorination agent, include typicallydimethylaminosulfur trifluoride, diethylaminosulfur trifluoride anddipropylaminosulfur trifluoride. Exemplary of thebis(dialkylamino)sulfur difluorides of formula (III') which may also beused as the fluorination agent are bis(dimethylamino)sulfur, difluorideand bis(diethylamino)sulfur difluoride. All of these compounds are knownfluorinating agents [see, "J. Org. Chem.", 40, No. 5, 574-578 (1975)].

The reaction between a compound of formula (II) and a fluorination agentof formula (III) or (III') may be carried out in a non-polar organicsolvent, for example, an aromatic hydrocarbon such as benzene, tolueneand xylene or chlorinated hydrocarbon such as chloromethane,dichloromethane, chloroform and carbon tetrachloride, or acetonitrile ata temperature in the range of 0°-100° C., preferably at room temperatureunder an anhydrous condition. The fluorination agent of formula (III) or(III') may be used in an amount of 5-20 moles per mole of the compoundof formula (II). An amine such as tertiary alkylamine or pyridine may bepresent as the acid binding agent in the reaction mixture.

After the completion of the di-fluorination of the 5-ketone group of thecompound of formula (II), the reaction solution is added to an aqueoussolution of an alkali metal carbonate or an alkali metal hydrogencarbonate, preferably an aqueous sodium hdyrogen carbonate, toneutralize the acidic matter. The resulting mixture so neutralized isextracted with chloroform and the extract is washed with water, driedand concentrated under a reduced pressure to remove the chloroform andto leave the N,O-protected 2,5-dideoxy-5,5-difluorostreptaminederivative of formula (IV) as a solid.

The amino-protecting groups (X, X') and the hydroxyl-protecting groups(Y, Y') remaining in the compound of formula (IV) may be removed by aknown deprotecting method. Thus, an amino-protecting group of thealkoxycarbonyl or aryloxycarbonyl-type may be removed by an alkalinehydrolysis, whereas an amino-protecting group of thearalkyloxycarbonyl-type may be removed by an alkaline hydrolysis or byreduction. An amino-protecting group of the sulfonyl-type may also beremoved in a known manner by treating the compound of formula (IV) withmetallic sodium in liquefied ammonia (see, for example, U.K. Patent No.1,555,661 and Japanese Patent Publication No. 29720/85). Ahydroxyl-protecting group of the acyl type (Y, Y') may be removed bytreatment with sodium methoxide in methanol or by hydrolysis in anaqueous solution of alkali metal carbonate or alkali metal hydroxidesuch as sodium carbonate and sodium hydroxide. In cases where thehydroxyl-protecting groups form a single hydroxyl-protecting group ofthe alkylidene, cycloalkylidene or tetrahydropyranylidene type, suchgroup may be removed by hydrolysis in the presence of an inorganic acid,an organic acid or a cation exchange resin of strongly acidic nature,e.g. a cation exchange resin having sulfonic acid groups (see, forexample, U.K. Patent No. 2,043,634B).

The removal of all the residual amino-protecting and hydroxyl-protectinggroups from the compound of formula (IV) results in the formation of thedesired compound of formula (Ia) according to this invention. Theisolation and purification of the compound of formula (Ia) may suitablybe effected by chromatography on a molecular sieve agent such asCM-Sephadex C-25 as eluted with aqueous ammonia in a gradient elutiontechnique.

Further, according to the fourth aspect of this invention, there isprovided a process for the production of 5-deoxy-5,5-difluorosisomicinor 5-deoxy-5,5-difluoronetilmicin represented by the formula ##STR37##wherein R¹ is a hydrogen atom or ethyl group, characterized in that theprocess comprises reacting an N,O-protected 5-keto derivative ofsisomicin or netilmicin represented by the general formula ##STR38##wherein X and X' respectively are amino-protecting groups which may bethe same or different; Y and Y' respectively are hydroxyl-protectinggroups which may be the same or different; and optionally X' taken withY' may form such a single carbonyl group which can protectsimultaneously the 3"-imino group and the 4"-hydroxyl group of thecompound of formula (V); and R¹ is a hydrogen atom or ethyl group, witha dialkylaminosulfur trifluoride of the formura ##STR39## wherein R² isan alkyl group of 1 to 4 carbon atoms, or with a bis(dialkylamino)sulfur difluoride of the formula

    R.sup.2 N--SF.sub.2 --NR.sup.2                             (III')

wherein R² is as defined above, or with a fluorination agent equivalentto the compound of formula (III) or (III') in a non-polar organicsolvent, to di-fluorinate the ketone group at the 5-position of the5-keto derivative of the formula (V), thereby producing an N,O-protectedderivative of 5-deoxy-5,5-difluorosisomicin or5-deoxy-5,5-difluoronetilmicin represented by the formula ##STR40##wherein X, X', Y, Y' and R¹ respectively have the same meanings asdefined for the general formula (V) above, and then removing theremaining amino-protecting groups (X, X') and the remaininghydroxyl-protecting groups (Y, Y') from the compound of the formula (VI)by a conventional method.

Also in the starting compound of the general formula (V) employed in theprocess according to the fourth aspect of this invention, all of theamino groups born by said compound have been protected by any known andappropriate amino-protecting groups (X, X'). The sorts of theamino-protecting groups used for this purpose may be same as theamino-protecting groups present in the starting compound of the formula(II) used in the process according to the third aspect of thisinvention. Further, the hydroxyl groups other than the 5-hydroxyl groupof the starting compound of the general formula (V) may have beenprotected by the hydroxyl-protecting groups (Y, Y') which are of thesame sort as those for the hydroxyl groups of the compound of thegeneral formula (II). Besides, it is possible that X' at the3"-methylamino group and Y' at the 4"-hydroxyl group of the compound ofthe formula (V) are linked to each other to form a single carbonyl groupand thereby constitute the form of a cyclic carbamate, whereby the3"-methylamino group and 4"-hydroxyl group can be protectedsimultaneously. Such a 3", 4"-N,O-protected derivative wherein the3"-methylamino group and the 4"-hydroxyl group have been converted intothe form of the cyclic carbamate may be provided by preparing fromsisomicin or netilmicin firstly such N-protected derivative thereofhaving benzyloxycarbonyl groups as the amino-protecting groups (X, X')but having the hydroxyl group unprotected, and then reacting saidN-protected derivative with sodium hydride in dimethylformamide (DMF)(see Referential Examples 11 and 13 given hereinafter).

In the process according to the fourth aspect of this invention, thedi-fluorination of the 5-ketone group of the compound of the formula (V)may again be effected by reacting said ketone group with thefluorinating compound of the formula (III) or formula (III') in the samemanner as in the process according to the third aspect of thisinvention. The resulting 5,5-difluorinated derivative of the formula(VI) is then subjected to conventional deprotection methods for removalof the amino-protecting groups (X, X') and the hydroxyl-protectinggroups (Y, Y') therefrom. In this way, there is afforded5-deoxy-5,5-difluorosisomicin or 5-deoxy-5,5-difluoronetilmicin of thegeneral formula (Ib') as desired.

According to the fifth aspect of this invention, there is provided aprocess for the production of 5-deoxy-5,5-difluoroseldomycin factor 3represented by the formula ##STR41## characterized in that the processcomprises reacting an N,O-protected 5-keto derivative of seldomycinfactor 3 represented by the formula ##STR42## wherein X is anamino-protecting group and Y is a hydroxyl-protecting group, withdialkylaminosulfur trifluoride of the formula ##STR43## wherein R² is analkyl group of 1 to 4 carbon atoms, or with a bis(dialkylamino)sulfurdifluoride of the formula

    R.sup.2 N--SF.sub.2 --NR.sup.2                             (III')

wherein R² is as defined above, or with a fluorination agent equivalantto the compound of formula (III) or (III') in a non-polar organicsolvent, to di-fluorinate the ketone group at the 5-position of the5-keto derivative of the formula (VII), thereby producing anN,O-protected derivative of 5-deoxy-5,5-difluoroseldomycin factor 3represented by the formula ##STR44## wherein X and Y respectively havethe same meanings as defined above, and then removing the remainingamino-protecting group (X) and the remaining hydroxyl-protecting group(Y) from the compound of the formula (VIII) by a conventional method.

Also in the starting compound of the general formula (VII) employed inthe process according to the fifth aspect to this invention, all of theamino groups born by this seldomycin compound have been protected by anyknown and appropriate amino-protecting group (X). The sort of theamino-protecting group available for this purpose may be same as theamino-protecting groups which are provided in the starting compound ofthe general formula (II) used in the process according to the thirdaspect of this invention. Besides, the hydroxyl groups other than the5-hydroxyl group of the starting compound of the general formula (VII)may also have been protected by the hydroxyl-protecting group (Y) whichis of the same sort as for the hydroxyl groups of the compound of thegeneral formula (II).

Also in the process according to the fifth aspect of this invention, thedi-fluorination of the 5-ketone group of the compound of the formula(VII) may be achieved by reacting said ketone group with thefluorinating compound of the formula (III) or formula (III') in the samemanner as in the process according to the third aspect of thisinvention. The 5,5-difluorinated derivative so obtained of the formula(VIII) is then subjected to conventional deprotection methods. Thus,there is obtained 5-deoxy-5,5-difluoroseldomycin factor 3 of the generalformula (Id) as desired.

Method for preparing the N,O-protected 5-ketonized derivatives of theformula (II), formula (V) and formula (VII) which are employed as thestarting compound in the aforesaid processes according to the third,fourth and fifth aspects of this invention will next be described.

This preparing method comprises a series of stages as detailed below.Thus, at first, there is employed as an initial material a4-O-(aminoglycosyl)- or 4,6-di-O-(aminoglycosyl)-2-deoxystreptaminerepresented by the foregoing general formula (A). Then, there isconducted a first stage where the amino-protecting groups (X, X') areintroduced into all the amino groups of the 2-deoxystreptaminederivative of the general formula (A). This first stage compriseseffecting the reaction for the introduction into the amino groups of thecompound of the formula (A) such a mono-valent amino-protecting groups,including typically an alkoxycarbonyl group such as tert-butoxycarbonyland tert-amyloxycarbonyl; a cycloalkyloxycarbonyl group such ascyclohexyloxycarbonyl; an aralkyloxycarbonyl group such asbenzyloxycarbonyl; a hydrolytically cleavable, substituted loweralkanoyl group such as trifluoroacetyl and o-nitrophenoxyacetyl; aphosphinothioyl group such as diphenylphosphinothioyl anddimethylphosphinothioyl; a phosphinyl group such as dephenylphosphinyl;or alternatively such a divalent amino-protecting groups which may bephthaloyl or may form a Schiff base with the amino group to beprotected.

The introduction of the amino-protecting group of these kinds may beconducted by employing an appropriate known reagent for introduction ofthe amino-protecting group which may be in the form of an acylatingagent such as an acid halide, acid azide, active ester, acid anhydrideand the like, according to the techniques known per se in theconventional synthesis of peptides or for protection of amino groups ofkanamycins. By choosing the quantity of the reagent for introduction ofthe amino-protecting group in a proportion of 1 to 2 moles per one aminogroup present in the molecule of the starting 2-deoxystreptaminederivative of the general formula (A) as the initial material, it ispossible to prepare such an amino-protected derivative of the startingcompound of the general formula (A) of which all the amino groupspresent have been protected.

Then, in such cases when the above-mentioned first stage is achieved soas to introduce an alkylsulfonyl group, aralkylsulfonyl group orarylsulfonyl group as the amino-protecting group to be used, this firststage may be effected in a known manner by reacting the startingcompound of the general formula (A) with a sulfonic acid halide of theformula

    R.sup.3 SO.sub.2 Hal

where R³ is a lower alkyl group, an aralkyl group, particularly benzylgroup or an aryl group, particularly phenyl group; and Hal is chlorineor bromine, preferably tosyl chloride in aqueous dioxane in the presenceof sodium carbonate at a temperature of 0° to 50° C. (see, for example,U.K. patent No. 1,555,661 and Japanese patent publication No.Sho-60-29720 specification).

Next, the second stage is conducted, where all of the hydroxyl groupsother than the 5-hydroxyl group of the above-mentioned amino-protectedderivative derived from the starting compound of the general formula (A)are protected by a hydroxyl-protecting group.

In this second stage, said amino-protected derivative is reacted withacetyl chloride or acetic anhydride in anhydrous pyridine at atemperature of 50° C. or less, e.g, at 0° C. with using acetyl chlorideor acetic anhydride in a proportion of 1.5 to 5 moles or in a slightlyexcessive proportion per one hydroxyl group present in saidamino-protected derivative. Then, all of the hydroxyl groups other thanthe 5-hydroxyl group of said amino-protected derivative, that is, allthe hydroxyl groups at the 2'-, 3'-, 4'-, 2"-, 3"- and 4"-positions andalso at the 6"-position (but excepting such a case where the hydroxylgroup is absent at the 6"-position) of the amino-protected derivativecan be protected by acetylation. In general, even when an acyl chloride,including a lower alkanoyl chloride other than acetyl chloride, as wellas an aroyl chloride such as benzoyl chloride is used in place of acetylchloride or acetic anhydride, all the hydroxyl groups other than the5-hydroxyl group of said amino-protected derivative can similarly beprotected by the acylation with the 5-hydroxyl group being not acylatedunder the action of the steric hindrance in the molecule of theamino-protected derivative. In this way, there is formed theN,O-protected 2-deoxystreptamine derivative having the free 5-hydroxylgroup and generically represented by the general formula (B) givenhereinbefore.

More particularly, the N,O-protected 2-deoxystreptamine derivative soprepared may include the N,O-protected 2-deoxystreptamine derivatives ofthe following general formulae (II'), (V') and (VII'):

(a) An N,O-protected derivative of kanamycin A, a deoxykanamycin A, adideoxykanamycin A, kanamycin B, a deoxykanamycin B, a dideoxykanamycinB, 3'-fluoro-3' -deoxykanamycin B, 3'-fluoro-3', 4'-dideoxykanamycin B,gentamicin C₁, gentamicin C_(1a) or gentamicin C₂ or sagamicinrepresented by the general formula ##STR45## wherein X and X'respectively are amino-protecting groups which may be the same ordifferent; Y is a hydroxyl-protecting group; A^(a) is a protectedhydroxyl group or a protected amino group; A^(b) and A^(c) respectivelyare a hydrogen atom, a protected hydroxyl group or a fluoro group; A^(d)is a hydrogen atom or methyl group, namely is same as A⁴ set forth inthe general formula (I) above; A^(e) is a hydrogen atom or methyl group,namely is same as A⁵ set forth in the general formula (I); and B^(a) isa hydrogen atom or methyl group, namely is same as B¹ set forth in thegeneral formula (I); B^(b) and B^(c) respectively are a hydrogen atom, aprotected hydroxyl group (OY) or a protected hydroxyl group (OY') asprotected by a different hydroxyl-protecting group, or a methyl group;B^(d) is a hydrogen atom or a hydroxymethyl group as protected at itshydroxyl moiety and having formula (--CH₂ OY); and Y' is ahydroxyl-protecting group different from the group Y.

(b) An N,O-protected derivative of sisomicin or netilmicin representedby the general formula ##STR46## wherein X and X' respectively areamino-protecting groups which may be the same or different; Y and Y'respectively are hydroxyl-protecting groups which may be the same ordifferent; and optionally X' taken with Y' may form such a singlecarbonyl group which can protect simultaneously the 3"-imino group andthe 4"-hydroxyl group of the compound of formula (V'); and R¹ is ahydrogen atom or ethyl group.

(c) An N,O-protected derivative of seldomycin factor 3 represented bythe formula ##STR47## wherein X is an amino-protecting group and Y is ahydroxyl-protecting group.

Further, the third stage is conducted, where the 5-hydroxyl group of thecompound of the general formula (B) is oxidized and thereby saidcompound is converted into the 5-keto derivative of the general formula(C) shown hereinbefore. Thus, more particularly, this third stagecomprises either oxidizing the 5-hydroxyl group of the N,O-protectedderivative of the above general formula (II') to produce the 5-ketoderivative of the general formula (II), or oxidizing the 5-hydroxylgroup of the N,O-protected derivative of the general formula (V') toproduce the 5-keto derivative of the general formula (V), or oxidizingthe 5-hydroxyl group of the N,O-protected derivative of the generalformula (VII') to produce the 5-keto derivative of the general formula(VII). The reaction for oxidation in this third stage may be conductedby the following procedure. Thus, said N,O-protected derivative isreacted e.g., with pyridinium chlorochromate (PCC) or a mixture ofdimethylsulfoxide-acetic anhydride as a mild oxidizing agent which isknown to oxidize a hydroxyl substitutent of organic compounds into aketone group, so that the oxidation of the 5-hydroxyl group into the5-ketone group can be achieved. Suitable examples of solvents availablein this oxidation reaction include dichloromethane, pyridine, DMSO, amixture of pyridine and DMSO, benzene, carbon tetrachloride, chloroform,acetonitrile and the like. The oxidation may suitably be carried out ata reaction temperature of -20° C. to 100° C. This oxidation reaction canbe finished within one week (see Referential Example 1(c) givenhereinafter).

According to the sixth aspect of this invention, there is provided aprocess for the production of a1-N-(α-hydroxyl-ω-aminoalkanoyl)-4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptaminederivative represented by the general formula ##STR48## wherein A¹, A²,A³, A⁴ and A⁵, as well as B¹, B², B³ and B⁴ have the same meanings asdefined for the general formula (I) hereinbefore or for the generalformula (Ia) hereinbefore, and n is an integer of 1 to 3, whichcomprises reacting the 1-amino group of a4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamine derivativerepresented by the general formula ##STR49## wherein A¹, A², A³, A⁴ andA⁵, as well as B¹, B², B³ and B⁴ respectively have the same meanings asdefined above, or the 1-amino group of such a protected derivative ofthe compound of formula (Ia) whose some or all of the amino groups otherthan said 1-amino group has or have been protected by amino-protectinggroups, with an α-hydroxy-ω-aminoalkanoic acid having the generalformula ##STR50## wherein n is an integer of 1 to 3, or such a protectedderivative of said aminoalkanoic acid whose the amino group has beenprotected by an amino-protecting group, and then removing from theresulting 1-N-acylated product the remaining amino-protecting groupswhen the latter protective groups exist, to produce the compound offormula (Ic).

In the process according to the sixth aspect of this invention, all orsome of the amino groups other than the 1-amino group of the2,5-dideoxy-5,5-difluorostreptamine derivative of the general formula(Ia) are protected by appropriate amino-protecting groups. Theamino-protecting group available for this purpose includes conventionalamino-protecting groups. For instance, there may be employed analkoxycarbonyl group such as methoxycarbonyl, ethoxycarbonyl andtert-butoxycarbonyl; an aralkyloxycarbonyl group such asbenzyloxycarbonyl; and an acyl group such as phenoxycarbonyl, acetyl,trifluoroacetyl and benzoyl, as well as a sulfonyl group such as tosyl.The introduction of the amino-protecting group of these kinds may beachieved in a known manner by reacting the2,5-dideoxy-5,5-difluorostreptamine derivative of the general formula(Ia) with an appropriate known reagent for the introduction of theamino-protecting group, which may be in the form of an acylating agentsuch as an acid halide, acid azide, active ester, acid anhydride and thelike, by a conventional method known in the synthesis of peptides. Bychoosing the quantity of the reagent for the introduction of theamino-protecting group in a proportion of 0.5 to 6 moles per mole of thecompound of the formula (Ia), it is possible to prepare from thedifluoro compound of the general formula (Ia) different, partiallyamino-protected derivatives thereof in different proportions due todifference in the reactivities of the respective amino groups of thecompound of the formula (Ia).

In this process, it is possible to use, such amino,protected derivativeof the difluoro compounds of general formula (Ia) whose all or some ofthe amino groups other than the 1-amino group have been protected, forexample, a 3,2',6',3"-tetra-N-protected derivative, a3,2',6'-tri-N-protected derivative, a 6',3"-di-N-protected derivative ora 6'-mono-N-protected derivative. A mixture of two or more of thosepartially N-protected derivatives may also be used for the acylation ofthe 1-amino group with the substituted aminoalkanoic acid of the formula(IX) in the process of the sixth aspect of this invention.

The production of the 1-N-acylated product of the general formula (Ic)in a high yield in the process of the sixth aspect of this invention,can be achieved by acylating the 1-amino group of the starting difluorocompound of formula (Ia) selectively with the α-hydroxy-ω-amino-alkanoicacid of formula (IX). Accordingly, it will be evident that mostpreferably, such a protected derivative of the compound of formula (Ia)whose all the amino groups other than the 1-amino group have beenprotected, namely a3,2',6',3"-tetra-N-protected-2,5-dideoxy-5,5-difluorostreptaminederivative is employed as the starting substance to be 1-N-acylated inthis process.

To prepare such an amino-protected derivative of the compound of thegeneral formula (Ia) of which all the amino groups other than the1-amino group have been protected, it is convenient to utilize thefollowing procedures. Thus, for instance, firstly a 3,6'-di-N-protectedor 3,2',6'-tri-N-protected derivative derived from the2,5-dideoxy-5,5-difluorostreptamine derivative of the formula (Ia) isproduced in a high yield by application of such amino-protecting methodaccording to claim 1 of Japanese patent application first publication"Kokai" No. Sho-55-64598 or U.S. Pat. No. 4,297,485, that comprisesreacting the compound of the formula (Ia) with zinc cation to form azinc complex, reacting this zinc complex with the reagent for theintroduction of amino-protective group to protect with theamino-protective group all of the amino groups other than the two1-amino and 3"-amino groups of said zinc complex (with the 1-amino and3"-amino groups remaining blocked by complexing with zinc cation), andthen removing the zinc cation from the resulting amino-protected zinccomplex, e.g. by treatment with a cation-exchange resin or treatmentwith hydrogen sulfide or treatment with aqueous ammonia. (In this case,it is also possible to replace zinc cation by a cation of cobalt,copper, nickel and the like which is employed in the process of Japanesepatent application first publication "Kokai" No. Sho-52-153944 or itscorresponding U.S. Pat. No. 4,136,254). Subsequently, the 3"-amino groupof said 3,6'-di-N-protected or 3,2',6'-tri-N-protected derivative soproduced is preferentially acylated by application of a method forpreparation of an amino-protected derivative having selectively all theamino groups protected except the 1-amino group set forth in claim 15 ofJapanese patent application first publication "Kokai" No. Sho-55-164696or U.S. Pat. No. 4,297,485, whereby there can be prepared in a highyield such a tri-N-protected or tetra-N-protected derivative derivedfrom the 5,5-difluoro compound of formula (Ia), of which all the aminogroups other than the 1-amino group have been protected. The U.S. Pat.No. 4,297,485 specification describes such a process wherein such anamino-protected derivative of an aminoglycoside antibiotic whose all theamino groups other than the 1-amino and 3"-amino groups have beenprotected is reacted with e.g., with a formic acid ester, adi-halogenated alkanoic acid ester or a tri-halogenated alkanoic acidester for preferential acylation of the 3"-amino group, so that the3"-amino group can preferentially be protected by the formyl group,di-haloalkanoyl group or tri-haloalkanoyl group without involving theacylation of the 1-amino group of said aminoglycoside antibiotic.

In the process of the sixth aspect of this invention, theα-hydroxy-ω-aminoalkanoic acid of the formula (IX) used may be anα-hydroxy-ω-aminoalkanoic acid of which the amino group has beenprotected or not. This substituted alkanoic acid (IX) is used to acylatethe 1-amino group of the compound of the formula (Ia) or its partiallyamino-protected derivative. This 1-N-acylation reaction may be conductedby reacting the compound of formula (Ia) or its amino-protectedderivative with the α-hydroxy-ω-aminoalkanoic acid of formula (IX)according to a so-called dicyclohexylcarbodiimide method, mixed acidanhydride method, azide method, active ester method etc., at a reactiontemperature suitably in a range of 0° C. to 30° C. The amino-protectinggroup which may be used conveniently in this process of the presentinvention includes tert-butoxycarbonyl group andp-methoxybenzyloxycarbonyl group which are easily cleavable by treatmentwith aqueous trifluoroacetic or acetic acid or with dilute aqueoushydrochloric acid. Benzyloxycarbonyl group which is removable by aconventional hydrogenolysis in the presence of a catalyst of platinumgroup metal, such as palladium or platinum oxide is also a convenientamino-protecting group.

The 1-N-acylation in the process of the sixth aspect of this inventionmay desirably be carried out in an aqueous organic solvent according tothe active ester method. For example, N-hydroxysuccinimide ester of(S)-4-tert-butoxycarbonylamino-2-hydroxybutyric acid may preferably beused as the active ester which may be prepared by a conventional methodof preparing such active esters. This active ester may usually be usedin a proportion of from 1 to 3 molar equivalents and preferably of from1 to 1.5 molar equivalents per mole of the starting compound of formula(Ia) or a partially amino-protected derivative thereof to be1-N-acylated. The solvent used as the reaction medium may be awater-miscible organic solvent such as dioxane, dimethoxyethane,dimethylformamide, tetrahydrofuran, triethylamine and the like.

The 1-N-acylated product so obtained from the 1-N-acylation step is thensubjected to the removal of the amino-protecting groups, when theseamino-protective groups are still remaining in the 1-N-acylated product.The removal of the amino-protecting groups is effected by a conventionaldeprotecting technique. Thus, the amino-protecting group ofalkoxycarbonyl type is removed by acid hydrolysis with an aqueoussolution of trifluoroacetic acid or acetic acid etc., or with a diluteaqueous solution of an inorganic acid such as hydrochloric acid. Theamino-protecting group of aralkyloxycarbonyl type such asbenzyloxycarbonyl may easily be removed by an ordinary catalyticreduction (hydrogenolysis).

The synthesis of the compound of the formula (Ic) in accordance with theprocess of the sixth aspect of this invention is convenient to beconducted by a synthetic route comprising starting from an unprotectedcompound of the formula (Ia), for example, 5-deoxy-5,5-difluorokanamycinB, firstly preparing from such starting compound its amino-protectedderivative having all the amino groups protected except the 1-aminogroup by utilization of the amino-protecting method of U.S. Pat. No.4,297,485 and subsequently carrying out the process of the sixth aspectof this invention with using the so prepared amino-protected derivative.More concretely, for instance, it is convenient to conduct such asynthetic route comprising the following steps (i) to (iv).

(i) Firstly, the starting compound of formula (Ia) and zinc acetate (orcobalt acetate) are either suspended in dimethylsulfoxide (DMSO) ordissolved in a mixture of water and dimethylformamide (DMF), and theresulting suspension or solution of the complex of the compound (Ia) andzinc cation (or cobalt cation) as formed is reacted withN-(benzyloxycarbonyloxy)-succinimide (namely, a reagent for theintroduction of the amino-protecting benzyloxycarbonyl group) to protectthe 3-, (2'-) and 6'-amino groups of the compound (Ia) moiety of thezinc complex with the benzyloxycarbonyl groups, and thereby to form thecomplex of zinc (or cobalt) with such partially amino-protectedderivative of the compound (Ia), followed by removing the zinc (orcobalt) cation from the latter complex by treating with acation-exchange resin (such as Amberlite CG-50) , to give3,(2'),6'-tris(or bis)-N-benzyloxy-carbonylated compound (Ia), namelyCompound (a) ( . . . Step i).

(ii) Compound (a) is then reacted with ethyl trifluoroacetate in DMSO orDMF to protect the 3"-amino group of Compound (a) with thetrifluoroacetyl group, affording 3, (2'),6'-tris(orbis)-N-benzyloxycarbonyl-3"-N-trifluoro-acetylated compound (Ia), namelyCompound (b) ( . . . Step ii).

(iii) Further, Compound (b) is reacted withN-(benzyloxycarbonyl)-(S)-4-amino-2-hydroxybutyryloxy-succinimide orwith N-(benzyloxycarbonyl)-(S)- or(RS)-3-amino-2-hydroxypropionyloxysuccinimide in an aqueoustetrahydrofuran in the presence of sodium carbonate, so that the 1-aminogroup of Compound (b) is selectively acylated with the(S)-4-benzyloxycarbonylamino-2-hydroxybutyryl group or with the (S)- or(RS)-3-benzyloxy carbonylamino-2-hydroxypropionyl group ( . . . Stepiii). Thereby, there is formed 1-N-[(S)-4-(benzyloxycarbonyl)amino-2-hydroxybutyryl]- or 1-N-[(S)- or (RS)-3-(benzyloxy-carbonyl)amino-2-hydroxypropionyl]-3,(2'),6'-tris (orbis)-N-(benzyloxycarbonyl)-3"-N-trifluoroacetylated compound (Ia),namely Compound (c) as the 1-N-acylation product.

(iv) Compound (c) is then subjected to deprotecting treatment comprisingacidic or alkaline hydrolysis for the removal of the amino-protectingtrifluoroacetyl group therefrom and subsequent catalytic hydrogenolysisin the presence of a platinum group metal catalyst such as palladium orRaney nickel for the removal of the amino-protecting benzyloxycarbonylgroups therefrom ( . . . Step iv).

The compound of the formula (Ic) as desired may thus be produced with ahigh efficiency through these step (i) to step (iv) described above.

Furthermore, the compounds of the general formula (I) according to thisinvention also include a compound represented by the general formula##STR51## wherein (i) A² and A³ are each a hydroxyl group, or (ii) A² isa hydrogen atom and A³ is a hydroxyl group, or (iii) A² and A³ are eacha hydrogen atom, or (iv) A² is a fluoro group and A³ is a hydroxylgroup, or (v) A² is a fluoro group and A³ is a hydrogen atom, and whichis namely (i) 5-deoxy-5,5-difluoroneamine, or (ii)5,3'-dideoxy-5,5-difluoroneamine, or (iii)5,3',4'-trideoxy-5,5-difluoroneamine, or (iv)5,3'-dideoxy-5,5,3'-trifluoroneamine, or (v)5,3',4'-trideoxy-5,5,3'-trifluoroneamine. The compound of the generalformula (Ia-4) may be produced by hydrolysing the 5,5-difluorokanamycinB derivative having the general formula (Ia-2) given hereinbefore, witha diluted hydrochloric acid at a temperature of 50°-100° C. so that theglycosyl group linked to the 6-position of said kanamycin B compound(Ia-2) is cleaved therefrom (see Examples 19 and 20 given hereinafter).

Besides, an antibacterial composition comprising the compound of thisinvention having the general formula (I) as the active ingredient may beformulated by mixing the compound of the general formula (I) with one ormore carriers of various kinds and optionally further adding differentadditives into the composition.

The formulations for administration of the compound of the generalformula (I) according to this invention may be of any forms for oral,parenteral and intrarectal administrations. When an injectableformulation is to be prepared, the compound of the general formula (I)according to this invention or a salt thereof to be used as the activeingredient may be admixed with a pH-adjuster, buffer, stabilizer,excipient and the like, and the resulting admixture may be lyophilizedby a conventional method to prepare a lyophilized preparation for theinjection. Further, the compound of this invention may be admixed with apH-adjuster, buffer, stabilizer, isotonising agent, local anestheticsand the like, and the resulting mixture may be formulated in aconventional manner into a preparation adapted for subcutaneous,intramuscular or intravenous injection.

When a solid formulation for oral administration is to be prepared, thecompound of this invention may be mixed with excipient, optionallytogether with binder, disintegrator, lubricant, colorant, flavor,odor-improver and the like, followed by forming the resulting mixtureinto tablet, coated tablet, granule, powder, capsule and the like byconventional methods.

When a liquid formulation for oral administration is to be prepared, thecompound of this invention may be mixed with flavor, buffer, stabilizer,odor-improver and the like, followed by formulating the resultingmixture into a syrup or dried syrup by conventional methods.

Where a suppository formulation for intrarectal administration isprepared, the compound of this invention may be mixed with excipientand, if necessary, also with surfactant, followed by forming theresultant mixture into a suppository drug by a conventional method.

The dosage of the compound of this invention will depend on the natureof diseases to be treated and conditions of the diseases, but itsoptimum dosage can be decided by ordinary and appropriate, preliminarytests.

The production of the compound of the general formula (I) according tothis invention is next illustrated with reference to the followingReferential Examples and Examples, to which this invention is notlimited.

In the following Referential Examples and Examples, there are shownstructural formulae for representing the concerned compounds, wherein Acdenotes an abbreviation of acetyl group; Z denotes an abbreviation ofbenzyloxycarbonyl group; Bz denotes an abbreviation of benzoyl group;AFT denotes an abbreviation of trifluoroacetyl group.

Referential Example 1

(a) Preparation of 1,3,6,3"-tetrakis(N-benzyloxycarbonyl)kanamycin A(Compound 1) ##STR52##

Kanamycin A sulfate (3 g) and sodium carbonate (2.93 g) were dissolvedin a mixed solvent (60 ml) of water and acetone (1:1) to whichbenzyloxycarbonyl chloride (2.90 ml) was added, followed by effectingthe reaction for 3 hours under ice-cooling and stirring. Water (200 ml)was added to the reaction solution, and the precipitate formed wasfiltered, washed with water, dried and then washed with ethyl ether togive the titled Compound (1) (4.6 g, yield 90%).

(b) Preparation of 2',3',4',2",4",6"-hexa-O-acetyl-1,3,6',3"-tetrakis(N-benzyloxycarbonyl) kanamycin A (Compound 2) ##STR53##

The compound (1) (4.6 g) as obtained in the step (a) above was dissolvedin dry pyridine (92 ml) to which acetic anhydride (10.2 ml) was addedand O-acetylation reaction was conducted at room temperature overnight.Water (9.73 ml) was added to the reaction solution, and the resultingsolution was left to stand for 30 minutes and then concentrated todryness. The residue was extracted with chloroform and the resultantextract solution was successively washed with an 10% aqueous solution ofpotassium hydrogen sulfate, a saturated aqueous solution of sodiumhydrogen carbonate and water, dried over anhydrous sodium sulfate andconcentrated to dryness to obtain the titled compound (2) (5.66 g, yield99%).

[α]_(D) ²³ +71° (c 1.02, chloroform).

(c) Preparation of 2',3',4',2",4",6"-hexa-O-acetyl-1,3,6',3"-tetrakis(N-benzyloxycarbonyl)-5-deoxy-5-oxokanamycin A (Compound 3 ) ##STR54##

The compound (2) (893 mg) as obtained in the step (b) above, namely,2',3',4',2",4",6"-hexa-O-acetyl-1,3,6',3"-tetrakis(N-benzyloxycarbonyl)kanamycinA was dissolved in dry dimethylsufoxide (2.7 ml) to which aceticanhydride (1.8 ml) was added, followed by effecting the reaction at roomtemperature for 4 days. At this time, a part of dimethylsufoxide actedas an oxidizing agent and the OH group at the 5-position of the compound(2) was oxidized. The reaction solution was poured into a saturatedaqueous solution (90 ml) of sodium hydrogen carbonate and the resultantsolution was stirred for 2 hours. The precipitate formed was filtered,taken into chloroform and then successively washed with an saturatedaqueous solution of sodium hydrogen carbonate and water and dried overanhydrous sodium sulfate. The resultant solution was concentrated todryness to obtain the titled Compound (3) (749 mg, yield 84%).

Example 1

(a) Preparation of2',3',4',2",4",6"-hexa-O-acetyl-1,3,6',3"-tetrakis(N-benzyloxycarbonyl)-5-deoxy-5,5-difluorokanamycinA (Compound 4) ##STR55##

The compound (3) (551 mg) as obtained in Referential Example 1(c) wasdissolved in a dry dichloromethane (17 ml) to which diethylaminosulfurtrifluoride (0.74 ml) was added under ice-cooling, followed by effectingthe reaction at room temperature for 5 hours (for di-fluorination). Theresultant reaction solution was poured into an aqueous solution (60 ml)of sodium hydrogen carbonate with stirring and under ice-cooling, andthen stirring was continued for 1 hour. The dichloromethane layer wasseparated and concentrate to dryness. The resultant solid was separatedand purified by chromatography on silica gel column (developer:chloroform-acetone, 9:2 ) to obtain the titled Compound (4) (268 mg,yield 48%).

(b) Preparation of1,3,6',3"-tetrakis(N-benzyloxycarbonyl)-5-deoxy-5,5-difluorokanamycin A(Compound 5) ##STR56##

The compound (4) (185 mg) as obtained in the step (a) above wasdissolved in a mixed solvent of methanol (7.4 ml) and water (0.74 ml) towhich sodium carbonate (210 mg) was added, followed by vigorous stirringat room temperature for 2 hours. A dilute hydrochloric acid was added tothe reaction solution to neutralize it and the resultant solution wasconcentrated to dryness. The residue was washed with water and dried toobtain the titled Compound (5) (121 mg, yield 81%).

(c) Preparation of 5-deoxy-5,5-difluorokanamycin A (Compound 6)##STR57##

The compound (5) (73.4 mg) as obtained in the step (b) above wasdissolved in a mixed solvent of dioxane, acetic acid and water (4:1:1)(4.4 ml) to which palladium black was added as a catalyst, followed byeffecting the catalytic reduction at room temperature for 1 hour (forde-benzyloxycarbonylation). The reaction solution was filtered and thefiltrate was concentrated to dryness. The resulting solid waschromatographed in a column of "CM-Sephadex C-25" developed with aqueousammonia while changing the concentration of ammonia from 0N to 0.15N,and the fractions of the eluate containing the intended substance wereconcentrated to dryness to obtain the titled Compound (6) (23.6 mg,yield 58%, calculated as the monocarbonate monohydrate).

[α]_(D) ²⁰ +130° (c 1, water).

Referential Example 2

(a) 3,6'-bis (N-benzyloxycarbonyl)-5-deoxy-5,5-difluorokanamycin A(Compound 7) ##STR58##

The monocarbonate (87.2 mg) of the compound (6) as obtained in Example1(c) was suspended in dry dimethylsulfoxide (0.9 ml), to which zincacetate dihydrate (172 mg) was added, followed by stirring at 80° C. for1 hour. The resultant homogeneous solution was cooled to roomtemperature, to which N-(benzyloxycarbonyloxy)succinimide (136 mg) wasadded, followed by effecting the reaction at room temperature for 2hours (for introducing benzyloxycarbonyl group as the amino-protectinggroup).

Ethyl ether was added to the reaction solution, the depositedprecipitate was washed with ethyl ether and the resulting solid wasdissolved in a mixed solvent of dioxane and water (1:1). The solutionwas passed through a column of "Amberlite CG-50" (NH₄ ⁺ -form) resin forthe adsorption, and the resin column was developed gradiently with mixedsolvent of water and dioxane (1:1) containing ammonia, while changingthe concentration of ammonia from 0N to 0.1N. Zinc ion was not elutedand the fractions of the eluate containing the intended substance wereconcentrated to dryness to obtain the titled Compound (7) (96.8 mg,yield 81%).

(b)3,6'-bis(N-benzyloxycarbonyl)-5-deoxy-5,5-difluoro-3"-N-trifluoroacetylkanamycinA (Compound 8) ##STR59##

The compound (7) (105 mg) as obtained in the step (a) above wasdissolved in dry dimethylsulfoxide (0.5 ml) to which ethyltrifluoroacete (0.02 ml) was added, followed by effecting the reactionat room temperature for 1 hour (for selective trifluoroacetylation of3"-OH group). Ethyl ether was added to the reaction solution and thedeposited precipitate was washed with ethyl ether to obtain the titledCompound (8) (117 mg).

Example 2

(a) Preparation of3,6'-bis(N-benzyloxycarbonyl)-1-N-[(S)-4-benzyloxycarbonylamino-2-hydroxybutyryl]-5-deoxy-5,5-difluoro-3"-N-trifluoroacetylkanamycinA (Compound 9) ##STR60##

The compound (8) (112 mg) as obtained in Referential Example 2(b) wasdissolved in a mixed solvent (3.4 ml) of tetrahydrofuran and water(1:1). The resulting solution was mixed with sodium carbonate (11 mg)and then with the N-hydroxysuccinimide ester (26.5 mg) of(S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid dissolved intetrahydrofuran (1.7 ml). After reacting for 2 hours, 4 hours and 6hours at room temperature, N-hydroxysuccinimide ester of(S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid (18 mg) was furtheradded at each time. After effecting the reaction at room temperature for7 hours (for the (S)-4-protected-amino-2-hydroxybutyrylation of theamino group at 1-position), the reaction solution was concentrated andthe residue was washed with water, dried and washed with ethyl ether anddried to obtain the titled Compound (9) (135 mg).

(b) Preparation of1-N-[(S)-4-amino-2-hydroxybutyryl]-5-deoxy-5,5-difluorokanamycin A(Compound 10) ##STR61##

The compound (9) (350 mg) as obtained in the step (a) above wasdissolved in a mixed solvent (25 ml) of 2N aqueous ammonia andtetrahydrofuran (4:3) (two layers were formed), followed by effectingthe reaction for 1 day at 28° C. with stirring to remove the3"-N-trifluoroacetyl group. Thereafter, the reaction solution wasconcentrated and the resulting solid was dissolved in a mixed solvent ofdioxane (12 ml), water (2.5 ml) and acetic acid (2.5 ml). The solutionso obtained was subjected to catalytic reduction in the presence ofpalladium black as a catalyst at room temperature for 1 hour to removethe N-benzyloxycarbonyl group. After filtration of the reactionsolution, the filtrate was concentrated to obtain a solid. The solid wasdissolved in water and the solution was chromatographed gradiently in acolumn of "CM-Sephadex C-25" as developed with aqueous ammonia, whilechanging the concentration of ammonia from 0N to 0.5N. Fractions of theeluate containing the intended substance were concentrated to dryness toobtain the titled Compound (10) (103 mg).

[α]^(D) ²⁰ +48° (c 1, water)

Referential Example 3

(a) Preparation of1,3,6',3"-tetrakis(N-benzyloxycarbonyl)-3',4'-dideoxykanamycin A(Compound 11) ##STR62##

3',4'-dideoxykanamycin A (527 mg) and sodium carbonate (520 mg) wereadded to a mixed solvent (10.5 ml) of water and acetone (1:1) to whichbenzyloxy chloride (0.68 ml) was added, followed by stirring underice-cooling for 3 hours (for introducing the N-benzyloxycarbonyl group).Water (70 ml) was added to the reaction solution and the deposited solidwas filtered, washed with water, dried and washed with ethyl ether anddried to obtain the titled Compound (11) (957 mg, yield 83%).

(b) Preparation of2',2",4",6"-tetra-O-acetyl-1,3,6',3"-tetrakis(N-benzyloxycarbonyl)-3',4'-dideoxykanamycinA (Compound 12) ##STR63##

The Compound (11) (803 mg) as obtained in the step (a) above wasdissolved in dry pyridine (16 ml) to which acetic anhydride (1.38 ml)was added, followed by effecting the acetylation reaction overnight atroom temperature. Water (1.32 ml) was added to the reaction solution,and after allowing to stand for 30 minutes, the solution wasconcentrated to dryness. The resulting residue was extracted withchloroform, washed successively with 10% aqueous solution of potassiumhydrogen sulfate, a saturated aqueous solution of sodium hydrogencarbonate and water and then dried over anhydrous sodium sulfate. Theresulting solution was concentrated to dryness to afford the titledCompound (12) (841 mg, yield 90%).

(c) Preparation of 2',2",4",6"-tetra-O-acetyl-1,3,6',3"-tetrakis(N-benzyloxycarbonyl)-5,3',4'-trideoxy-5-oxokanamycin A(Compound 13) ##STR64##

The compound (12) (784 mg) as obtained in the step (b) above wasdissolved in dry dimethylsulfoxide (2.35 ml) to which acetic anhydride(1.57 ml) was added, followed by effecting the reaction for 3 days atroom temperature (for the oxidation of the hydroxyl group at5-position).

The reaction solution was poured into a saturated aqueous solution (80ml) of sodium hydrogen carbonate under ice-cooling and the solution wasstirred for 3 hours. The resulting precipitate was filtered, washed withwater and extracted with chloroform. The organic layer was washedsuccessively with a saturated aqueous solution of sodium hydrogencarbonate and water and then dried over anhydrous sodium sulfate. Theresulting solution was concentrated to dryness to obtain the titledCompound (13) (625 mg, 80% yield of crude product).

Example 3

(a) Preparation of2',2",4",6"-tetra-O-acetyl-1,3,6',3"-tetrakis(N-benzyloxycarbonyl)-5,3',4'-trideoxy-5,5-difluorokanamycin A (Compound 14) ##STR65##

The compound (13) (329 mg) as obtained in Referential Example 3(c) wasdissolved in dry dichloromethane (10 ml) to which diethylaminosulfurtrifluoride (0.49 ml) was added under ice-cooling, followed by effectingthe reaction for 7 hours at room temperature (for the di-fluorination).The reaction solution was poured into an aqueous solution (40 ml) ofsodium hydrogen carbonate under ice-cooling and the solution was stirredfor 30 minutes. The dichloromethane layer was separated and concentratedto dryness. The resulting solid was isolated and purified by subjectingto chromatography on silica gel column (developer: chloroform-acetone,9:2) to give the titled Compound (14) (137 mg, yield 41%).

(b) Preparation of1,3,6',3"-tetrakis(N-benzyloxycarbonyl)-5,3',4'-trideoxy-5,5-difluorokanamycinA (Compound 15) ##STR66##

The compound (14) (452 mg) as obtained in the step (a) above wasdissolved in a mixed solvent of methanol (18 ml) and water (1.8 ml) towhich sodium carbonate (570 mg) was added, followed by effecting thereaction for 2 hours at room temperature under vigorous stirring (forthe de-acetylation). Dilute hydrochloric acid was added to the reactionsolution to neutralize and the solution was concentrated to dryness. Theresulting residue was washed with water and then dried to obtain thetitled Compound (15) (294 mg, yield 76%).

(c) Preparation of 5,3',4'-trideoxy-5,5-difluorokanamycin A (Compound16) ##STR67##

The compound (15) (161 mg) as obtained in the step (b) above wasdissolved in a mixed solvent (9.7 ml) of dioxane, acetic acid and water(4:1:1), followed by effecting catalytic reduction for 3 hours at roomtemperature in the presence of palladium black as a catalyst (for thedeprotection). After filtration of the reaction solution, the filtratewas concentrated to dryness. The resulting solid was developed withaqueous ammonia, in a column of "CM-Sephadex C-25", while changing theconcentration of ammonia from 0N to 0.15N. Fractions of the eluatecontaining the intended substance were concentrated to dryness to obtainthe titled Compound (16) (47.7 mg, yield 54%, calculated as themonocarbonate monohydrate). [α]_(D) ²⁰ +130° (c 1, water)

Referential Example 4

Preparation of3,6'-bis(N-benzyloxycarbonyl)-5,3',4'-trideoxy-5,5-difluorokanamycin A(Compound 17) ##STR68##

The monocarbonate (121 mg) of the compound (14) as obtained in Example3(c) above was suspended in dry dimethylsufoxide (1.2 ml) to which zincacetate dihydrate (252 mg) was added, followed by stirring for 1 hour at80° C. The resulting homogeneous solution was cooled to room temperatureand N-(benzyloxycarbonyl)succinimide (201 mg) was added, followed byeffecting the reaction for 1.5 hours at room temperature. Ethyl etherwas added to the reaction solution and the deposited precipitate waswashed with ethyl ether. The resulting solid was dissolved in a mixedsolvent of water and dioxane (2:3) and the solution was chromatographedgradiently in "Amberlite CG-50" (NH₄ ⁺ -form) resin with a mixed solventof water and dioxane (2:3) containing ammonia, while changing theconcentration of ammonia from 0N to 0.1N. Zinc ions were not eluted andthe fractions of the eluate containing the intended substance wereconcentrated to dryness to obtain the titled Compound (17) (131 mg,yield 79%).

Example 4

(a) Preparation of1-N-[(S)-4-amino-2-hydroxybutyryl]-5,3',4'-trideoxy-5,5-difluorokanamycinA (Compound 18) ##STR69##

3"-N-Trifluoroacetylation and1-N-[(S)-4-benzyloxycarbonylamino-2-hydroxybutyryl]ation,de-3"-N-trifluoroacetylation and de-tris-N-benzyloxycarbonylation of theCompound (17) as obtained in the Referential Example 4 were effectedaccording to the same manner as in Referential Example 2(b) and Example2(a) and (b) to afford the titled Compound (18).

[α]_(D) ²⁰ +105° (c 1, water)

Referential Example 5

(a) Preparation of3',4',2",4",6"-penta-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)kanamycinB (Compound 19 ) ##STR70##

1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)kanamycin B (1.05 g) wasdissolved in dry pyridine (21 ml) to which acetic anhydride (1.72 ml)was added, followed by effecting the O-acetylation reaction overnight atroom temperature.

Water (1.64 ml) was added to the reaction solution, the solution wasleft to stand for 30 minutes at room temperature and then concentratedto dryness. The residue was extracted with chloroform and the chloroformextract was washed successively with 10% aqueous solution of potassiumhydrogen sulfate, a saturated aqueous solution of sodium hydrogencarbonate and water and then dried over anhydrous sodium sulfate. Theresulting solution was concentrated to dryness to obtain the titledCompound (19) (1.20 g, yield 97%).

(b) Preparation of3',4',2",4",6"-penta-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-5-deoxy-5-oxokanamycinB (Compound 20) ##STR71##

The Compound (19) (1.10 g) as obtained in the step (a) above wasdissolved in dry dimethylsulfoxide (3.3 ml) to which acetic anhydride(2.2 ml) was added, followed by effecting the reaction for 4 days atroom temperature (for oxidation of the hydroxyl group at 5-position).The reaction solution was poured in small portions into a saturatedaqueous solution of sodium hydrogen carbonate (110 ml) under ice-coolingand with stirring, and the solution was stirred for 2.5 hours underice-cooling. The resulting precipitate was filtered and washed withwater. The resulting solid was extracted with chloroform and washedsuccessively with a saturated aqueous sodium hydrogen carbonate andwater and then dried over anhydrous sodium sulfate. The resultingsolution was concentrated to dryness to give the titled Compound (20)(1.03 g, yield 94%).

Example 5

(a) Preparation of3',4',2",4",6"-penta-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-5-deoxy-5,5-difluorokanamycinB (Compound 21) ##STR72##

The Compound (20) (876 mg) as obtained in Referential Example 5(b) wasdissolved in dry dichloromethane (26 ml) to which diethylaminosulfurtrifluoride (1.1 ml) was added under ice-cooling, followed by effectingthe di-fluorination reaction for 6 hours at room temperature. Thereaction solution was poured into an aqueous solution (110 ml) of sodiumhydrogen carbonate under ice-cooling and the solution was stirred for 30minutes. The dichloromethane layer was separated and the aqueous layerwas extracted with chloroform three times. The combined organic layerwas washed successively with a saturated aqueous solution of sodiumhydrogen carbonate and water and then dried over anhydrous sodiumsulfate. The organic layer was concentrated to dryness. The resultingsolid was isolated and purified by subjecting to chromatography onsilica gel column (developer: chloroform-acetone, 9:2) to obtain thetitled Compound (21) (403 mg, yield 45%).

(b) Preparation of1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-5-deoxy-5,5-difluorokanamycinB (Compound 22) ##STR73##

The Compound (21) (688 mg) as obtained in the step (a) above wasdissolved in a mixed solvent (14 ml) of dry tetrahydrofuran and drymethanol (1:1), to which a 1N solution (0.34 ml) of sodium methoxide inmethanol was added, followed by effecting the reaction for 30 minutes atroom temperature (for de-acetylation). Dilute hydrochloric acid wasadded to the reaction solution to neutralize and the solution wasconcentrated to dryness. The resulting residue was washed with water toafford the titled Compound (22) (568 mg, yield 97%).

(c) Preparation of 5-deoxy-5,5-difluorokanamycin B (Compound 23)##STR74##

The Compound (22) (408 mg) as obtained in the step (b) above wasdissolved in a mixed solvent of dioxane (14 ml), acetic acid (2.8 ml)and water (3.5 ml), followed by effecting the catalytic reduction for 3hours at room temperature in the presence of palladium black as acatalyst (for the deprotection). After filtration of the reactionsolution, the filtrate was concentrated to dryness. The resulting solidwas chromatographed on a column of "CM-Sephadex C-25" as developed withaqueous ammonia while changing the concentration of ammonia from 0N to0.15N. Fractions of the eluate containing the intended substance wereconcentrated to dryness to obtain the titled Compound (23) (131 mg,yield 65%, calculated as the monocarbonate monohydrate).

Example 6

(a) Preparation of1-N-[(S)-4-amino-2-hydroxybutyryl]-5-deoxy-5,5-difluorokanamycin B(Compound 24 ) ##STR75##

3,2',6'-Tris-N-benzyloxycarbonylation and 3'-N-trifluoroacetylation,1-N-[(S)-4-benzyloxycarbonylamino-2-hydroxybutyryl]ation,de-3"-N-trifluoroacetylation and de-tetrakis-N-benzyloxycarbonylation ofthe Compound (23) as obtained in Example 5(c) were effected according tothe same manner as in Referential Example 2(a) and (b) and also inExample 2(a) and (b) to obtain the titled Compound (24).

Referential Example 6

(a) Preparation of 1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)tobramycin(Compound 25) ##STR76##

Tobramycin sulfate (1.29 g) was dissolved in a mixed solvent (26 ml) ofwater and acetone (1:1) to which sodium carbonate (1.2 g) andbenzyloxycarbonyl chloride (1.13 ml) were added, followed by stirringfor 3 hours under ice-cooling. Water (130 ml) was added to the reactionsolution and the resulting precipitate was filtered, washed with waterand dried and washed with ethyl ether to obtain the titled Compound (25)(2.00 g, yield 97%).

[α]_(D) ²⁴ +53° (c 2.0, pyridine)

(b) Preparation of4',2",4",6"-tetra-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)tobramycin(Compound 26) ##STR77##

The Compound (25) (2.09 g) as obtained in the step (a) above wasdissolved in dry pyridine (42 ml), to which acetic anhydride (3.47 ml)was added, followed by effecting the O-acetylation overnight at roomtemperature.

Water (3.3 ml) was added to the reaction solution and after allowing tostand for 30 minutes, the solution was concentrated to dryness. Theresidue was extracted with chloroform and the extract solution waswashed successively with 10% aqueous solution of potassium hydrogensulfate, a saturated aqueous solution of sodium hydrogen carbonate andwater and then dried over anhydrous sodium sulfate. The resultingsolution was concentrated to dryness to obtain the titled Compound (26)(2.40 g, yield 100%).

¹ H-NMR spectrum (in deutero-pyridine-D₂ O): δ1.88, 2.00, 2.05, and2.18, each 3H singlet(acetyl)

(c) Preparation of 4',2",4",6"-tetra-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-5-deoxy-5-oxotobramycin (Compound27) ##STR78##

Pyridinium chlorochromate (4.75 g) and powdered Molecular Sieve 3A (5.5g) were suspended in dry dichloromethane (24 ml), to which was added asolution in dry dichloromethane (280 ml) of the Compound (26) (2.40 g)obtained in the above step (b), followed by stirring at room temperaturefor 3 days to oxidize the hydroxyl group at the 5-position.

The insoluble matter in the reaction solution was filtered with aid ofCelite and washed with chloroform. The filtrate and the washings werecombined and the resulting solution was concentrated to 200 ml andwashed successively with 10% aqueous solution of potassium hydrogensulfate and a saturated aqueous solution of sodium hydrogen carbonate.The solution was then dried over anhydrous sodium sulfate andconcentrated to dryness. The resulting residue was purified bychromatography on silica gel column (developer: chloroform-acetone, 7:2)to obtain the titled Compound (27) (1.82 g, yield 76%).

[α]_(D) ²⁴ +65° (c 2, CHCl₃) ¹ H-NMR spectrum (in deutero-pyridine):δ1.85, 1.91, 1.95, and 1.99, each 3H singlet(acetyl).

Example 7

(a) Preparation of4',2",4",6"-tetra-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-5-deoxy-5,5-difluorotobramycin(Compound 28) ##STR79##

The Compound (27) (1.69 g) as obtained in Referential Example 6(c) wasdissolved in dry dichloromethane (50 ml) to which diethylaminosulfurtrifluoride (2.06 ml) was added under ice-cooling, followed by effectingthe reaction for 8 hours at room temperature. The reaction solution waspoured into a saturated aqueous solution (200 ml) of sodium hydrogencarbonate under ice-cooling and the solution was stirred for 30 minutes.The dichloromethane layer was when separated, washed with a saturatedaqueous solution of sodium hydrogen carbonate and then dried overanhydrous sodium sulfate. The solution was concentrated to dryness andthe resulting residue was purified by chromatography on silica gelcolumn (developer: chloroform-acetone, 4:1) to afford the titledCompound (28) (1.13 g, yield 66%).

[α]_(D) ²⁴ +67° (c 2, CHCl₃). ¹ H-NMR spectrum (in deutero-pyridine):δ1.79(s,3H), 1.97(s,6H), 2.00(s,3H), each, methyl of acetyl group.

¹⁹ F-NMR spectrum (in deutero-pyridine, CFCl₃ internal standard):δ-110.3(d, J_(F),F' =250 Hz) and -129.5(dt, J_(F),H-4 =J_(F),H-6 =19 Hz)

(b) Preparation of 1,3,2',6',3"-pentakis(N-benzyloxycarbonyl-5-deoxy-5,5-difluorotobramycin (Compound29) ##STR80##

The Compound (28) (400 mg) as obtained in the step (a) above wasdissolved in a mixed solvent of dry tetrahydrofuran (4 ml) and drymethanol (3.8 ml) to which a 1N solution (0.2 ml) of sodium methoxide inmethanol was added, followed by effecting the reaction for 40 minutes atroom temperature. Dilute hydrochloric acid was added to the reactionsolution to neutralize and the solution was concentrated to dryness. Theresulting residue was washed with water to obtain the titled Compound(29) (347 mg, yield 99%).

[α]_(D) ²³ +70° (c 2, pyridine)

¹⁹ F-NMR spectrum (in deutero-pyridine, CFCl₃ internal standard):δ-111.1(d, J_(F),F' =250 Hz) and -129.0 (dt, J_(F),H-4 =J_(F),H-6 =19Hz)

(c) Preparation of 5-deoxy-5,5-difluorotobramycin (Compound 30)##STR81##

The Compound (29) (942 mg) as obtained in the step (b) above wasdissolved in a mixed solvent of dioxane (32 ml), acetic acid (8.1 ml)and water (6.5 ml), followed by effecting the catalytic reduction for 2hours at room temperature in the presence of palladium black as acatalyst. After filtration of the reaction solution, the filtrate wasconcentrated to dryness. The resulting solid was dissolved in water andwas chromatographed in a column of "CM-Sephadex C-25" developed withaqueous ammonia, while changing the concentration of ammonia from 0N to0.15N. Fractions of the eluate containing the intended substance wereconcentrated to dryness to obtain the titled Compound (30) (409 mg,yield 89%, calculated as the monocarbonate monohydrate).

[α]_(D) ²² +134° (c 1, H₂ O)

¹⁹ F-NMR spectrum (in 20% ND₃ /D₂ O; CCl₃ F external standard):δ-110.6(d, J_(F),F' =246 Hz) and -128.6(d, J_(F),H-4 =J_(F),H-6 =21 Hz)

Referential Example 7

(a) Preparation of3,2',6'-tris(N-benzyloxycarbonyl)-5-deoxy-5,5-difluorotobramycin(Compound 31) ##STR82##

In dry dimethylsulfoxide (3.4 ml) was suspended the monocarbonate (335mg) of 5-deoxy-5,5-difluorotobramycin, namely Compound (30) as obtainedin Example 6(c), followed by addition of zinc acetate dihydrate (678mg). The resultant mixture was stirred at 80° C. for 1 hour and theresulting homogeneous solution was cooled to room temperature.N-(benzyloxycarbonyloxy)succinimide (558 mg) was added portionwise tosaid solution which was then subjected to the reaction for 1 hour atroom temperature.

Ethyl ether was added to the reaction solution, and a precipitate asdeposited was washed with ethyl ether. The solid obtained was repeatedlywashed with 3N aqueous ammonia to remove the zinc cation therefrom. Theresulting product was washed with water and then dried to give 537 mg ofthe titled Compound (31).

(b) Preparation of3,2',6'-tris(N-benzyloxycarbonyl)-5-deoxy-5,5-difluoro-3"-N-trifluoroacetyltobramycin(Compound 32) ##STR83##

Compound (31) (537 mg) as obtained in the step (a) above was dissolvedin dry dimethylsulfoxide (2.7 ml), followed by addition of ethyltrifluoroacetate (0.094 ml). The reaction was made at room temperaturefor 1 hour. Ethyl ether was added to the reaction solution, and theprecipitate as obtained was washed repeatedly with ethyl ether anddried, thereby affording the titled Compound (32) as a solid (596 mg).

Example 8

(a) Preparation of3,2',6'-tris(N-benzyloxycarbonyl)-1-N-[(S)-4-benzyloxycarbonylamino-2-hydroxybutyryl]-5-deoxy-5,5-difluoro-3"-N-trifluoroacetyltobramycin(Compound 33) ##STR84##

Compound (32) (62.1 mg) as obtained in Referential Example 7(b) wasdissolved in a mixed solvent (1.9 ml) of tetrahydrofuran and water(1:1). The resulting solution was mixed with sodium carbonate (5.3 mg)and then with a solution of N-hydroxysuccinimide ester (26.5 mg) of(S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid in tetrahydrofuran (1ml), followed by further addition of N-hydroxysuccinimide ester (6.6 mg)of (S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid at roomtemperature each 2 hours, 4 hours and 6 hours later. The reaction wasconducted at room temperature for 7 hours. Thereafter, the reactionsolution was concentrated to a small volume and the residue was washedwith water. After drying, the residue was washed with ethyl ether anddried to afford the titled Compound (33) as a solid (68.3 g).

(b) Preparation of1-N-[(S)-4-amino-2-hydroxybutyryl]-5-deoxy-5,5-difluorotobramycin(Compound 34) ##STR85##

Compound (33) (68.3 mg) as obtained in the step (a) above was dissolvedin a (4:3) mixed solvent (3.4 ml) of 2N aqueous ammonia andtetrahydrofuran to afford a mixture comprising two layers. The reactionmixture was agitated overnight at 28° C. to remove the3"-N-trifluoroacetyl group from Compound (33). Thereafter, the reactionsolution was concentrated to dryness and the resulting solid residue wasdissolved in a mixed solvent of dioxane (2 ml), water (0.5 ml) andacetic acid (0.4 ml). The resulting solution was subjected to catalyticreduction with hydrogen gas at room temperature for 1 hour in thepresence of palladium black as a catalyst to remove theN-benzyloxycarbonyl groups from Compound (33). The reaction solution wasfiltered and the filtrate was concentrated to dryness. The resultingsolid was dissolved in water and was chromatographed gradiently in acolumn of "CM-Sephadex C-25" as developed with aqueous ammonia as aneluent while changing the concentration of ammonia from 0N to 0.5N.Fractions of the eluate containing the intended compound were collectedand concentrated to dryness, giving the titled Compound (34) as a solid(21.6 mg).

Specific rotation: [α]_(D) ²³ +89° (c 3, H₂ O)

¹⁹ F-NMR Spectrum (in 20% ND₃ -D₂ O; CCl₃ F external standard):δ-110.3(d, J_(F),F' =247 Hz) and -129.5(dt, J_(F),H-4 =J_(F'H-6) =20 Hz)

Referential Example 8

(a) Preparation of1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-3',4'-dideoxykanamycin B(Compound 35) ##STR86##

A mixture of 3',4'-dideoxykanamycin B (1.01 g) and sodium carbonate (1.2g) was dissolved in a mixed solvent (20 ml) of water and acetone (1:1),followed by addition of benzyloxycarbonyl chloride (1.61 ml) underice-cooling and stirring.

One hour later, the reaction mixture was warmed to room temperature,followed by addition of water (130 ml). The resulting precipitate wascollected by filtration, washed with water and then dried under reducedpressure. The precipitate was further washed with ethyl ether and driedto afford the titled Compound (35) as a solid (2.35 g). Yield: 94%

(b) Preparation of2",4",6"-tri-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-3',4'-dideoxykanamycinB (Compound 36) ##STR87##

Compound (35) (1.73 g) as obtained in the step (a) above was dissolvedin dry pyridine (35 ml), to which acetic anhydride (3 ml) was added toconduct the reaction overnight at room temperature. The reactionsolution was mixed with water (2.9 ml) under ice-cooling, allowed tostand at room temperature for 30 minutes and then concentrated todryness under reduced pressure. The resulting residue was extracted withchloroform and the extract solution was washed successively with a 10%aqueous solution of potassium hydrogen sulfate, a saturated aqueoussolution of sodium hydrogen carbonate and water and dried over anhydroussodium sulfate. This solution was concentrated to dryness to obtain asolid (1.95 g) . The resultant solid was purified by columnchromatography on silica gel as developed with a mixed solvent ofchloroform and ethanol (30:1) as eluent to afford the titled Compound(36) as a solid (1.79 g). Yield: 93%

(c) Preparation of 2",4",6"-tri-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-5,3',4'-trideoxy-5-oxokanamycin B (compound 37)##STR88##

Compound (36) (1.60 g) as obtained in the step (b) above was dissolvedin dry dimethylsulfoxide (4.8 ml), followed by addition of aceticanhydride (3.2 ml). The reaction was conducted at room temperature for aperiod of 3 days. The reaction solution was added portionwise to asaturated aqueous solution of sodium hydrogen carbonate (160 ml) and theresulting precipitate was filtered, washed with water and dried. Thesolid so obtained was then purified by column chromatography on silicagel as developed with a mixed solvent of chloroform and ethanol (30:1)as eluent to give the titled Compound (37) as a solid (1.18 g). Yield:74%

Example 9

(a) Preparation of2",4",6"-tri-O-acetyl-1,3,2',6'3"-pentakis(N-benzyloxycarbonyl)-5,3',4'-trideoxy-5,5-difluorokanamycinB (Compound 38) ##STR89##

Compound (37) (264 mg) as obtained in Referential Example 8(c) wasdissolved in dry dichloromethane (7.9 ml), followed by addition ofdiethylaminosulfur trifluoride (0.36 ml) under ice-cooling. The reactionwas made at room temperature for a period of 5 hours. The reactionsolution was then poured into a saturated aqueous solution of sodiumhydrogen carbonate (35 ml) under ice-cooling and stirring and maintainedfor 30 minutes with stirring. The dichloromethane layer obtained wasseparated from the resulting reaction solution and the aqueous layer wasextracted three times with chloroform. The chloroform extract wascombined with the dichloromethane layer and the combined solution waswashed successively with a saturated aqueous solution of sodium hydrogencarbonate and water, and dried over anhydrous sodium sulfate. The driedsolution was concentrated to dryness and the resulting solid wassubjected to column chromatography on silica gel as developed with amixed solvent of chloroform and acetone (9:2) as eluent to conduct theseparation and purification of the reaction product, thereby affordingthe titled Compound (38) as a solid (130 mg). Yield: 49%

¹ H-NMR Spectrum (in deutero-chloroform): δ1.79, 1.88, 1.99 (eachsinglet, 3H) (methyl portion of acetyl group),

¹⁹ F-NMR Spectrum (in deutero-chloroform; CCl₃ F internal standard);δ-110.4 (broad, doublet, J_(F),F' =247 Hz), -129.6 (broad, doublet)

(b) Preparation of 1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-5,3',4'-trideoxy-5,5-difluorokanamycin B (Compound39) ##STR90##

Compound (38) (129 mg) as obtained in the step (a) above was dissolvedin a mixed solvent of dry tetrahydrofuran (1.3 ml) and anhydrousmethanol (1.3 ml), to which a methanolic solution (0.065 ml) of 1Nsodium methoxide was added, followed by effecting the de-acetylationreaction at room temperature for 30 minutes. The reaction solution wasneutralized by addition of dilute hydrochloric acid and concentrated todryness. The residue obtained was washed with water and dried so thatthere was afforded the titled Compound (39) as a solid (116 mg). Theyield was stoichiometric.

¹⁹ F-NMR Spectrum (in deutero-pyridine, CCl₃ F internal standard):δ-110.9 (doublet, J_(F),F' =250 Hz), δ-129.0 (broad, double triplet)

(c) Preparation of 5,3',4'-trideoxy-5,5-difluorokanamycin B (Compound40) ##STR91##

Compound (39) (2.55 g) as obtained in the step (b) above was dissolvedin a mixed solvent of dioxane (88 ml), acetic acid (17 ml) and water (22ml), and the resulting solution was subjected to catalytic reduction atroom temperature for 4 hours in the presence of palladium black as acatalyst. The reaction solution was filtered and the filtrate wasconcentrated to dryness to give a solid. The solid was dissolved inwater and the resulting solution was passed through a column of"CM-Sephadex C-25" which was then eluted gradiently with aqueous ammoniawhile changing the concentration of ammonia from 0N to 0.15N. The activefractions of the eluate containing the intended compound were combinedand concentrated to dryness to afford the titled Compound (40) as asolid (953 mg) (Yield: 77%, calculated as themonocarbonate-monohydrate).

Specific rotation: [α]_(D) ²¹ +144° (c 2.0, H₂ O)

¹⁹ F-NMR Spectrum (in 20% aqueous deutero-ammonia, CCl₃ F internalstandard): δ-110.0(d, J_(F),F' =246 Hz) and -128.8(dt, J_(F),H-4=J_(F),H-6 =21 Hz)

Referential Example 9

(a) Preparation of3,2',6'-tris(N-benzyloxycarbonyl)-5,3',4'-trideoxy-5,5-difluorokanamycinB (Compound 41) ##STR92##

In dry dimethylsulfoxide (8.3 ml) was suspended the monocarbonate (830mg) of 5,3',4'-trideoxy-5,5-difluorokanamycin B, namely Compound (40)obtained in Example 9(c), followed by addition of zinc acetate dihydrate(1.75 g). The resultant mixture was stirred at 80° C. for 1 hour and theresulting homogeneous solution was cooled to room temperature, followedby addition in four portions of N-(benzyloxycarbonyloxy)succinimide(1.39 g) to said solution. The reaction mixture was subjected to thereaction for 1 hour at room temperature.

Ethyl ether was added to the reaction solution and a precipitated solidobtained was repeatedly washed with 3N aqueous ammonia to remove thezinc cation from the reaction product. The resulting solid product waswashed with water and dried to give the titled Compound (41) as a solid(1.36 g).

(b) Preparation of3,2',6'-tris(N-benzyloxycarbonyl)-5,3',4'-trideoxy-5,5-difluoro-3"-N-trifluoroacetylkanamycinB (Compound 42) ##STR93##

Compound (41) (1.36 g) as obtained in the step (a) above was dissolvedin dry dimethylsulfoxide (6.8 ml), followed by addition of ethyltrifluoroacetate (0.24 ml). The reaction was conducted at roomtemperature for 1 hour. Ethyl ether was added to the reaction solution,and the precipitate obtained was washed repeatedly with ethyl ether anddried, thereby affording the titled compound (42) as a solid (1.50 g).

Example 10

(a) Preparaion of3,2',6'-tris(N-benzyloxycarbonyl)-1-N-[(S)-4-benzyloxycarbonylamino-2-hydroxybutyryl]-5,3',4'-trideoxy-5,5-difluoro-3"-N-trifluoroacetylkanamycinB (Compound 43) ##STR94##

Compound (42)(1.5 g) as obtained in Referential Example 9(b) above wasdissolved in a mixed solvent (45 ml) of tetrahydrofuran and water (1:1),followed by addition of sodium carbonate (130 mg). The resultingsolution was then mixed with a solution of the N-hydroxysuccinimideester (650 mg) of (S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid intetrahydrofuran (23 ml). To the reaction mixture were added successivelythe N-hydroxysuccinimide ester (218 mg) of(S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid and sodium carbonate(66 mg) at room temperature each 2 hours, 4 hours, 6 hours and 8 hourslater. The reaction was conducted at room temperature for 9 hours.Thereafter, the reaction solution was concentrated to a small volume andthe resulting residue was washed with water. After drying, the residuewas washed with ethyl ether and dried to give the titled Compound (43)as a solid (1.77 g).

(b) Preparation of1-N-[(S)-4-amino-2-hydroxybutyryl]-5,3',4'-trideoxy-5,5-difluorokanamycinB (Compound 44) ##STR95##

Compound (43) (1.77 g) as obtained in the step (a) above was suspendedin a mixed solvent (124 ml) of 2N aqueous ammonia and tetrahydrofuran(4:3), followed by effecting the reaction at room temperature for 4 dayswith stirring to remove the 3"-N-trifluoroacetyl group from Compound(43). Thereafter, the reaction solution was concentrated to dryness andthe resulting solid was dissolved in a mixed solvent of dioxane (63 ml),acetic acid (13ml) and water (16 ml). The resulting solution wassubjected to catalytic reduction at room temperature for 3 hours in thepresence of palladium black as a catalyst to remove theN-benzyloxycarbonyl groups from Compound (43).

The reaction solution obtained was filtered and the filtrate wasconcentrated to dryness to give a solid. The solid was dissolved inwater and the solution was passed through a column of "CM-Sephadex C-25"which was then developed gradiently with aqueous ammonia while changingthe concentration of ammonia from 0N to 0.5N. Fractions of the eluatecontaining the intended compound were combined and concentrated todryness to afford the titled Compound (44) as a solid (612 mg).

Specific rotation: [α]_(D) ²¹ +92° (c 3.0, H₂ O)

¹⁹ F-NMR Spectrum (in 20% ND₃ -D₂ O, CCl₃ F external standard):δ-110.4(d, J_(F),F =247 Hz) and -129.6(dt, J_(F),H-4 =J_(F),H-6 =˜19 Hz)

Referential Example 10

(a) Preparation of1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-4'-deoxykanamycin B (Compound45) ##STR96##

4'-Deoxykanamycin B (821 mg) was dissolved in water (8.2 ml), in whichsodium carbonate (950 mg) was then added and dissolved. To this solutionwas added acetone (8.2 ml), followed by addition of benzyloxycarbonylchloride (1.3 ml) under ice-cooling and stirring. The reaction wasconducted for 3 hours under ice-cooling and stirring.

The reaction solution was warmed to room temperature and admixed withwater (100 ml). The precipitate thus formed was filtered, washed withwater and dried, and subsequently the resultant residue was washed withethyl ether and dried to give the titled Compound (45) as a solid (1.76g). Yield: 88%

(b) Preparation of3',2",4",6"-tetra-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-4'-deoxykanamycinB (Compound 46) ##STR97##

Compound (45) (1.63 g) as obtained in the step (a) above was dissolvedin dry pyridine (33 ml), followed by addition of acetic anhydride (2.4ml). The O-acetylation reaction was made overnight at room temperature.Under ice-cooling, the reaction solution was admixed with water (2.3ml), allowed to stand at room temperature for 30 minutes and thenconcentrated to dryness. The resulting residue was extracted withchloroform, and the extract solution was successively washed with a 10%aqueous solution of potassium hydrogen sulfate, a saturated aqueoussolution of sodium hydrogen carbonate and water and dried over anhydroussodium sulfate. The solution obtained was concentrated to dryness,thereby affording the titled Compound (46) as a solid (1.70 g). Yield:91%

(c) Preparation of 3',2",4",6"-tetra-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-5,4'-dideoxy-5-oxokanamycin B (Compound47) ##STR98##

Compound (46) (1.54 g) as obtained in the step (b) above was dissolvedin dry dimethylsulfoxide (4.6 ml) and the resultant solution was admixedwith acetic anhydride (3.1 ml) to effect the reaction at roomtemperature for 3 days (for the oxidation of 5-OH group). The reactionsolution obtained was poured with stirring into an ice-cooled saturatedaqueous solution of sodium hydrogen carbonate (150 ml) and maintainedfurther for 3 hours under agitation. A solid precipitated was filteredand washed with water. The solid was extracted with chloroform, and theresultant solution in chloroform was successively washed with asaturated aqueous solution of sodium hydrogen carbonate and water andthen dried over anhydrous sodium sulfate. The solution was concentratedto dryness to give the titled Compound (47) as a solid (1.38 g). Yield:90%

Example 11

(a) Preparation of3',2",4",6"-tetra-O-acetyl-1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-5,4'-di-difluorokanamycin B (Compound 48) ##STR99##

Compound (47) (993 mg) as obtained in Referential Example 10(c) wasdissolved in dry dichloromethane (30 ml), followed by addition ofdiethylaminosulfur trifluoride (1.31 ml) under ice-cooling to conductthe reaction at room temperature for 5 hours. The reaction solution waspoured with stirring into an ice-cooled saturated aqueous solution ofsodium hydrogen carbonate (110 ml) and maintained for 30 minutes withstirring. The resulting dichloromethane layer was separated from thereaction solution and concentrated to dryness to give a solid. The solidobtained was subjected to column chromatography on silica gel as elutedwith a mixed eluent of chloroform and acetone (9:2) to conduct theseparation and purification of the intended compound, thereby affordingthe titled Compound (48) as a solid (414 mg). Yield: 41%

(b) Preparation of1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)-5,4'-dideoxy-5,5-difluorokanamycinB (Compound ##STR100##

Compound (48) (307 mg) as obtained in the step (a) above was dissolvedin a mixed solvent (6 ml) of dry tetrahydrofuran and anhydrous methanol(1:1), followed by addition of a methanolic solution (0.15 ml) of 1Nsodium methoxide to effect the de-acetylation reaction at roomtemperature for 30 minutes.

The reaction solution was neutralized by addition of dilute hydrochloricacid and concentrated to dryness. The resultant residue was washed withwater and dried to give the titled Compound (49) as a solid (265 mg).Yield: 99%

(c) Preparation of 5,4'-dideoxy-5,5-difluorokanamycin B (Compound 50)##STR101##

Compound (49) (244 mg) as obtained in the step (b) above was dissolvedin a mixed solvent of dioxane (8.4 ml), acetic acid (1.7 ml) and water(2 ml) and the resultant solution was subjected to catalytic reductionin the presence of palladium black as a catalyst at room temperature for2 hours. The reaction solution was filtered and the filtrate wasconcentrated to dryness. Thus obtained was passed through a column of"CM-Sephadex C-25" as eluted gradiently with aqueous ammonia whilechanging the concentration of ammonia from 0N to 0.15N. The activefractions of the eluate containing the intended product wereconcentrated to dryness to give the titled Compound (50) as a solid(73.8 mg). (Yield: 62%, calculated as the monocarbonate-monohydrate)

Example 12

Preparation of1-N[(S)-4-amino-2-hydroxybutyryl]-5,4'-dideoxy-5,5-difluorokanamycin B(Compound 51) ##STR102##

Compound (50) as obtained in Example 11(c) was subjected to3,2',6'-tris-N-benzyloxycarbonylation, 3"-N-trifluoroacetylation,1-N-[(S)-4-benzyloxycarbonylamino-2-hydroxybutyryl]ation,de-3"-N-trifluoroacetylation and thende-tetrakis-N-benzyloxycarbonylation in the similar manners to those inReferential Example 2(a) and (b) and also Example 2(a) and (b), therebyaffording the titled Compound (51) as a solid.

Referential Example 11

(a) Preparation of 1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)gentamicinC₁ (compound 52) ##STR103##

A mixture of gentamicin C₁ (1.30 g) and sodium carbonate (1.30 g) wassuspended in a mixed solvent of water (12 ml) and dioxane (25 ml),followed by addition of benzyloxycarbonyl chloride (2.30 ml) at 0° to 5°C. so as to conduct the reaction at room temperature for 1 hour. Thereaction solution was concentrated under reduced pressure, and theresultant residue was dissolved in chloroform, washed successively witha 5% aqueous solution of sodium hydrogen carbonate and water, dried overanhydrous magnesium sulfate and concentrated under reduced pressure. Theresulting solid residue was washed with isopropyl ether and dried sothat there was afforded the titled Compound (52) as a solid (3.25 g).Yield: 97%

(b) Preparation of1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonylgentamicin C₁(Compound 53) ##STR104##

Compound (52) (3.25 g) as obtained in the step (a) above was dissolvedin DMF (30 ml), followed by addition of sodium hydride (400 mg) at 0° to5° C. so as to effect the reaction at room temperature for 1 hour (forthe formation of 3",4"-N,O-carbonyl group, namely formation of cycliccarbamate group). The reaction solution was admixed with chloroform (50ml) and water (50 ml) and neutralized by addition of 1N hydrochloricacid with stirring. The chloroform layer formed was separated from thereaction solution, washed with water and concentrated under reducedpressure. The resulting residue was subjected to column chromatographyon silica gel eluting with a mixed eluent of chloroform and methanol(100:1) for the separation and purification of the intended product toafford the titled Compound (53) as a solid (2.68 g). Yield: 96%

(c) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonylgentamicinC₁ (Compound 54) ##STR105##

Compound (53) (2.67 g) as obtained in the step (b) above was dissolvedin pyridine (30 ml), followed by addition of benzoyl chloride (1.2 ml:4.4 mole equivalents) at 0° to 5° C. so as to effect the reaction atroom temperature for 1 hour (for the benzoylation of 2"-hydroxyl group).Water (1 ml) was added to the reaction solution which was thenconcentrated under reduced pressure. The resultant residue was dissolvedin chloroform and washed successively with a 5% aqueous solution ofsodium hydrogen carbonate and water. The solution in chloroform wasdried over anhydrous magnesium sulfate and concentrated under reducedpressure to give the titled Compound (54) as a solid (2.88 g; Yield %).

(d) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonyl-5-deoxy-5-oxogentamicinC₁ (Compound 55) ##STR106##

Compound (54) (2.9 g) as obtained in the step (c) above was dissolved inmethylene chloride (40 ml), followed by addition of "Molecular Sieve 4A"(2.80 g) and pyridinium chlorochromate (1.40 g: 2.6 mole equivalents).The resulting reaction solution was refluxed under heating for 5 hours(for the oxidation of 5-hydroxyl group). The reaction solution was thensubjected to column chromatography on silica gel as eluted with a mixedeluent of chloroform and methanol (25:1), thereby affording the titledCompound (55) as a solid (2.68 g). Yield: 93%

Example 13

(a) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonyl-5-deoxy-5,5-difluorogentamicinC₁ (Compound 56) ##STR107##

Compound (55) (2.68 g) as obtained in Referential Example 11(d) wasdissolved in methylene chloride (30 ml), followed by addition ofdiethylaminosulfur trifluoride (3.2 ml) under ice-cooling to conduct thereaction at room temperature for 8 hours (for the di-fluorination). Thereaction solution was subjected to thin layer chromatography (TLC) onsilica gel as eluted with a mixed eluent of chloroform and methanol(25:1), which showed the main product at Rf 0.54 and many by-products atRf 7.5 to 8.0. The reaction solution was admixed with a 5% aqueoussolution of NaHCO₃ (30 ml) and stirred for 30 minutes. The methylenechloride layer was separated from the reaction solution, washed withwater and concentrated under reduced pressure. The resulting residue wassubjected to column chromatography on silica gel as eluted with a mixedsolvent of chloroform and methanol (100:1) for the separation andpurification of the intended product, thereby affording the titledCompound (56) as a solid (1.56 g). Yield: 57%

(b) Preparation of 5-deoxy-5,5-difluorogentamicin C₁ (Compound 57)##STR108##

To liquefied ammonia (about 50 ml) at -50° to -60° C., was added asolution (about 2.5 ml) in tetrahydrofuran (THF) of Compound (56) (1.56g) as obtained in the step (a) above. Metal sodium (about 400 mg) wasadded to the resulting solution. The reaction was conducted for 10minutes. The resulting reaction solution was subjected to TLC on silicagel as eluted with the lower phase of a mixed solvent ofchloroform-methanol-28% aqueous ammonia (1:1:1), when the main productappeared at Rf 0.76. The reaction solution was concentrated to a smallvolume and admixed with water (10 ml) to conduct the reaction at 80° C.for 3 hours (for the removal of the carbamate ring). The resultingreaction solution was neutralized by addition of 6N hydrochloric acidand then adsorbed on a column of "CM-Sephadex C-25" (NH₄ ⁺ -form) resin.The column was washed with water and eluted with aqueous ammonia whilechanging the concentration of ammonia from 0N to 0.2N. The activefractions of the eluate were combined and concentrated under reducedpressure. The resultant residue was subjected to column chromatographyon silica gel as eluted with a mixed eluent of chloroform-methanol-28%aqueous ammonia (9:4:1) for the separation and purification of theintended product, thereby giving the titled Compound (57) as a solid(2.88 g). Yield: 45%

Specific rotation: [α]_(D) ²⁴ +147° (c 0.5, water)

¹ H-NMR (20% ND₃ /D₂ O): δ0.27 (3H, d, H-7', J_(6'),7' =7 Hz), 2.23 (3Hs, 4"-CH₃), 3.33 and 3.57(each 3H, s, 6'-NCH₃ and 6 12(each 1H, d, H-1'and H-1", J_(1"),2" =3.5 Hz)

¹⁹ F-NMR(20% ND₃ /D₂ O): δ-128.28(dt, J_(F),F =246 Hz, J₄,F =J₆,F =20Hz) and -113.28(dt, J₄,F =J₆,F =3.5 Hz)

Referential Example 12

(a) Preparation of3,2',6'-tris(N-benzyloxycarbonyl)-5deoxy-5,5-difluorogentamicin C₁(Compound 58) ##STR109##

Compound (57) (50.1 mg) as obtained in Example 13(b) and cobalt acetatetetrahydrate (78.0 mg) were dissolved in DMSO (1 ml) to prepare solutioncontaining a complex of Compound (57) with cobalt cation. The resultingsolution was admixed with benzyloxycarbonyloxy succinimide (78.0 mg) toconduct the reaction at room temperature for 1.5 hours. The reactionsolution was washed with ethyl ether, admixed with ethyl acetate (4 ml),washed with 28% aqueous ammonia and concentrated under reduced pressureto afford the titled Compound (58) as a solid (93.1 mg).

(b) Preparation of3,2',6'-tris(N-benzyloxycarbonyl)-5-deoxy-5,5-difluoro-3"-N-trifluoroacetylgentamicinC₁ (Compound 59) ##STR110## wherein AFT means a trifluoroacetyl grouphere and hereinafter.

Compound (58) (93.1 mg) as obtained in the step (a) above was dissolvedin dioxane (2 ml), followed by addition of ethyl trifluoroacetate (0.1ml) to conduct the reaction at room temperature for 1 hour (for thetrifluoroacetylation of 3"-amino group). The reaction solution wassubjected to TLC as eluted with a mixed solvent ofchloroform-methanol-28% aqueous ammonia (5:1:1), when a single spotappeared at Rf 0.73. The reaction solution was concentrated underreduced pressure to give the titled Compound (59) as a solid (101 mg).

Example 14

(a) Preparation of3,2',6'-tris(N-benzyloxycarbonyl)-1-N-[(S)-4-benzyloxycarbonylamino-2-hydroxybutyryl]-5-deoxy-5,5-difluoro-3"-N-trifluoroacetylgentamicinC₁ (Compound 60) ##STR111##

Compound (59) (101 mg) obtained in Referential Example 12(b) wasdissolved in dioxane (4 ml). The resultant solution was added withtriethylamine (0.5 ml) and the N-hydroxysuccinimide ester (the activeester) (47.5 mg) of (S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid,followed by conducting the reaction at room temperature for 2 hours. Theresulting reaction solution was concentrated under reduced pressure,followed by addition of water to form a solid. The resulting solid waswashed with water and then dried under reduced pressure to afford 126 mgof the titled Compound (60) as a solid.

(b) Preparation of1-N-[(S)-4-amino-2-hydroxybutyryl]-5-deoxy-5,5-difluorogentamicin C₁(Compound 61) ##STR112##

Compound (60) (126 mg) as obtained in the step (a) above was dissolvedin dioxane (2 ml), to which 28% aqueous ammonia (1 ml) was added,followed by conducting the reaction at room temperature for 16 hours(for the de-trifluoroacetylation). After concentrating the reactionsolution under reduced pressure, the resulting residue was dissolved ina mixed solvent of dioxane (2 ml), water (0.5 ml) and acetic acid (0.2ml) and then was subjected to catalytic reduction at ambient temperaturefor 2 hours under atmospheric pressure. The resulting reaction solutionwas filtered and the filtrate was concentrated to a small volume underreduced pressure, and then chromatographed in a column of "CM-SephadexC-25" (NH₄ ⁺ -form) as eluted gradiently with aqueous ammonia, whilechanging the concentration of ammonia continuously from 0N to 0.5N.Fractions of the eluate containing the desired compound were combinedtogether and concentrated to dryness to afford 23.9 mg (yield: 40% basedon Compound (57)) of the titled Compound (61) as a solid.

[α]_(D) ²⁴ +129° (c 12, water)

¹ H-NMR(DCl/D₂ O, pD<1): δ1.28(3H, d, H-7', J_(6'),7' =7 Hz) 1.40(3H, s,4"-CH₃), 2.78 and 2.92 (each 3H, s, 6'-NCH₃ and 3"-NCH₃), 5.18 and 5.74(each 1H, d, H-1' H-1", J_(1'),2' =J_(1"),2" =3.5 Hz)

¹³ C-NMR(DCl/D₂ O, pD<1): δ74.2 and 77.6 (each t, C-4 and C-6), 120.4(t, C-5)

Referential Example 13

(a) Preparation of 1,3,2',6',3"-pentakis (N-benzyloxycarbonyl)gentamicinC₂ (Compound 62) ##STR113##

Gentamicin C₂ (1.00 g) and sodium carbonate (1.0 g) were suspended in amixed solvent of water (10 ml) and dioxane (20 ml), followed by additionof benzyloxycarbonyl chloride (1.7 ml) at 0°-5° C. The resulting mixturewas treated in the same manner as in Referential Example 11(a) to afford2.93 g (yield: 98%) of the titled Compound (62) as a solid.

(b) Preparation of1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonylgentamicin C₂(Compound 63) ##STR114##

Compound (62) (1.63 g) as obtained in the step (a) above was dissolvedin DMF (16 ml). The resulting solution was added with sodium hydride(200 mg) at 0°-5° C, followed by conducting the reaction at roomtemerature for 1 hour (for the formation of the cyclic carbamate group).The resulting reaction solution was thereafter treated in the samemanner as in Referential Example 11(b) to afford 1.37 g (yield: 93%) ofthe titled Compound (63) as a solid.

(c) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonylgentamicinC₂ (Compound 64) ##STR115##

Compound (63) (716 mg) as obtained in the step (b) above was dissolvedin pyridine (7.2 ml), to which benzoyl chloride (0.35 ml, 5.7 moleequivalents) was added at 0°-5° C., followed by conducting the reactionat room temperature for 1 hour (for the benzoylation of the 2"-hydroxylgroup). The resulting reaction solution was thereafter treated in thesame manner as in Referential Example 11(c) to afford 776 mg (yield:98%) of the titled Compound (64) as a solid.

(d) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonyl-5-deoxy-5-oxogentamicinC₂ (Compound 65) ##STR116##

Compound (64) (1.32 g) as obtained in the step (c) above was dissolvedin methylene chloride (28 ml). The resulting solution was added with"Molecular Sieves 4A" (1.36 g) and pyridinium chlorochromate (960mg)(3.9 mole equivalents), followed by conducting the oxidative reactionunder reflux for 5 hours while heating. The reaction solution showed asingle spot at Rf value of 0.45 in silica gel TLC (a developing solvent:CHCl₃ -MeOH, 25:1). The resulting reaction solution was isolated andpurified by column chromatography on silica gel (a developing solvent:chloroform-methanol, 25:1) to afford 1.19 g (yield: 90%) of the titledCompound (65) as a solid.

Example 15

(a) Preparation of 2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonyl-5-deoxy-5,5-difluorogentamicinC₂ (Compound 66 ) ##STR117##

Compound (65) (1.19 g) as obtained in Referential Example 13(d) wasdissolved in methylene chloride (20 ml), to which diethylaminosulfurtrifluoride (1.6 ml) was added under ice-cooling, followed by conductingthe reaction at room temperature for 8 hours (for the di-fluorination).The reaction solution showed a single spot of the main product at Rfvalue of 0.47 and multiple spots of by-products at Rf of 0.74-0.77 insilica gel TLC (the TLC was developed with a mixed solvent of CHCl₃-MeOH, 25:1) . The resulting reaction solution was added with a 5%aqueous solution of sodium hydrogen carbonate (20 ml) and then stirredfor 30 minutes. Thereafter, the methylene chloride layer was separated,washed with water and then concentrated. The resulting residue waspurified by column chromatography on silica gel (developed with a mixedsolvent of chloroform-methanol, 25:1) to afford 755 mg (yield: 62%) ofthe titled Compound (66) as a solid.

(b) Preparation of 5-deoxy-5,5-difluorogentamicin C₂ (Compound 67)##STR118##

A solution (about 3 ml) in THF of Compound (66) of the step (2) (755 mg)was added into liquefied ammonia (about 30 ml) at -50° to -60° C. Theresulting mixture was added with metal sodium (about 400 mg), followedby conducting the reaction for 10 minutes. The resulting reactionsolution showed a spot of the main product at Rf value of 0.73 in silicagel TLC (the TLC was developed with the lower layer of a mixeddeveloping solvent of CHCl₃ -MeOH-28% NH₄ OH, 1:1:1). The reactionsolution was concentrated, added with water (10 ml) and then subjectedto the reaction for 3 hours to effect the breakdown of the cycliccarbamate group. The resulting reaction solution showed a spot of themain product at Rf value of 0.39 in silica gel TLC (the TLC wasdeveloped with the lower layer of a mixed developing solvent of CHCl₃-MeOH-28% NH₄ OH, 1:1:1).

The resulting reaction solution was neutralized with 6N hydrochloricacid, and then was treated with the resin "CM-Sephadex C-25 (NH₄ ⁺-form)" to adsorb the resultant reaction product therein. After washingthe resin column with water, the column was eluted gradiently withaqueous ammonia, while changing the concentration of ammonia from 0N to0.2N. Active fractions of the eluate containing the desired compoundwere concentrated and thereafter the resulting residue was purified bycolumn chromatography on silica gel, in such way that the column wasdeveloped with a developing solvent of CHCl₃ -MeOH-28% NH₄ OH (9:4:1),thereby affording 201 mg (yield: 58%) of the titled Compound (67) as asolid.

[α]_(D) ²⁴ +162° (c 0.4, water)

¹ H-NMR(20% ND₃ /D₂ O): δ1.80(3H, d, H-7', J_(6'),7' =7 Hz), 1.95(3H, s,4"-CH₃), 3.28(3H, s, NCH₃), 5.80 and 5.83 (each 1H, d, H-1' and H-1",J_(1'),2' =J_(1"),2" =3.5 Hz)

¹⁹ F-NMR(20% ND₃ /D₂ O): δ-133.13(dt, J_(F),F =246 Hz, J₄,F =J₆,F =21Hz) and -110.32(dt, J₄,F =J₆,F =4 Hz)

Referential Example 14

(a) Preparation of3,2',6'-tris(N-benzyloxycarbonyl)-5-deoxy-5,5-difluorogentamicin C₂(Compound 68) ##STR119##

Compound (67) (31.0 mg) as obtained in Example 15(b) and cobalt acetatetetrahydrate (51.5 mg) were dissolved in DMSO (0.6 ml). The resultingsolution was added with benzyloxycarbonyloxysuccinimide (49.1 mg),followed by conducting the reaction at room temperature for 1.5 hours.The resulting reaction solution was thereafter treated in the samemanner as in Referential&l Example 12(a) to afford 54.5 mg of the titledCompound (68) as a solid.

(b) Preparation of3,2',6'-tris(N-benzyloxycarbonyl)-5-deoxy-5,5-difluoro-3"-N-trifluoroacetylgentamicinC₂ (Compound 69) ##STR120##

Compound (68) (54.5 mg) as obtained in the step (a) above was dissolvedin dioxane (1 ml). The resulting solution was added with ethyltrifluoroacetate (0.1 ml), followed by conducting the reaction for 1hour at room temperature (for the trifluoroacetylation of the 3"-aminogroup). The resulting reaction solution was treated in the same manneras in Referential Example 12(b) to afford 60.5 mg of the titled Compound(69) as a solid.

Example 16

(a) Preparation of3,2',6"-tris(N-benzyloxycarbonyl)-1-N-[(S)-4-benzyloxycarbonylamino-2-hydroxybutyryl]-5-deoxy-5,5-difluorogentamicinC₂ (Compound 70) ##STR121##

Compound (69) (60.5 mg) as obtained in Referential Example 14(b) wasdissolved in dioxane (3 ml). The resultant solution was added withtriethylamine (0.4 ml) and N-hydroxysuccinimide (the active ester) (29.6mg) of (S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid, and thereaction was conducted at room temperature for 2 hours. The resultingreaction solution was thereafter treated in the same manner as inExample 14(a) to afford 74.5 mg of the titled Compound (70) as a solid.

(b) Preparation of1-N-[(S)-4-amino-2-hydroxybutyryl]-5-deoxy-5,5-difluorogentamicin C₂(Compound 71) ##STR122##

Compound (70) (74.5 mg) as obtained in the step (a) above was dissolvedin dioxane (1.5 ml). The resulting solution was added with 28% aqueousammonia (0.7 ml), followed by conducting the reaction at roomtemperature for 16 hours. The resulting reaction solution was thereaftertreated and subjected to the catalytic reduction and further treated inthe same manner as in Example 14(b), to afford 16.1 mg (yield: 43% basedon Compound (67)) of the titled Compound (71) as a solid.

[α]_(D) ²⁴ +107° (c 12, water)

¹ H-NMR(DCl/D₂ O, pD<1): δ1.34(3H, d, H-7', J_(6'),7' =7 Hz), 1.40(3H,s, 4"-CH₃), 2.93(3H, s, NCH₃), 5.16 and 5.70 (each 1H, d, H-1' and H-1",J_(1'),2' =J_(1"),2" =3.5 Hz)

¹³ C-NMR(DCl/D₂ O, pD<1): δ74.5 (each t, C-4 and C-6), 120.4 (t, C-5)

Referential Example 15

(a) Preparation of 1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)gentamicinC_(1a) (Compound 72) ##STR123##

Gentamicin C_(1a) (1.05 g) and sodium carbonate (1.0 g) were suspendedin a mixed solvent of water (10 ml) and dioxane (20 ml), to whichbenzyloxycarbonyl chloride (1.8 ml) was added at 0°-5° C., followed byconducting the reaction at room temperature for 1 hour. The resultingreaction solution was treated in the same manner as in ReferentialExample 11(a) to afford 2.58 g (yield: 99%) of the titled Compound (72)as a solid.

(b) Preparation of1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonylgentamicinC_(1a) (Compound 73) ##STR124##

Compound (72) (2.58 g) as obtained in the step (a) above was dissolvedin DMF (30 ml), to which sodium hydride (300 mg) was added at 0°-5° C.,followed by conducting the reaction at room temperature for 1 hour. Theresulting reaction solution was thereafter treated in the same manner asin Referential Example 11(b) to afford 2.27 g (yield: 97%) of the titledCompound (73) as a solid.

(c) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonylgentamicinC_(1a) (Compound 74) ##STR125##

Compound (73) (2.27 g) of the step (b) above was dissolved in pyridine(30 ml), to which benzoyl chloride (1.1 ml, 4.6 mole equivalents) wasadded at 0°-5° C., followed by conducting the reaction at roomtemperature for 1 hour. The resulting reaction solution was thereaftertreated in the same manner as in Referential Example 11(c) to afford2.37 g (yield: 95%) of the titled Compound (74) as a solid.

(d) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonyl-5-deoxy-5-oxogentamicinC_(1a) (Compound 75) ##STR126##

Compound (74) (2.37 g) as obtained in the step (c) above was dissolvedin methylene chloride (50 ml). The resulting solution was added with"Molecular Sieves 4A" (2.0 g) and pyridinium chlorochromate (1.30 g, 3.2mole equivalents), followed by conducting the oxidative reaction underreflux for 5 hours while heating. The reaction solution showed a singlespot at Rf value of 0.42 in silica gel TLC (the TLC was developed with amixed solvent of CHCl₃ -MeOH, 25:1). The resulting reaction solution wasisolated and purified by column chromatography on silica gel, in suchway that the column was developed with a mixed solvent of CHCl₃ -MeOH(25:1), thereby affording 2.23 g (yield: 94%) of the titled Compound(75) as a solid.

Example 17

(a) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonyl-5-deoxy-5,5-difluorogentamicinC_(1a) (Compound 76) ##STR127##

Compound (75) (2.23 g) as obtained in Referential Example 15(d) wasdissolved in methylene chloride (45 ml), added with diethylaminosulfurtrifluoride (3.0 ml) under ice-cooling, followed by conducting thereaction at room temperature for 8 hours (for the di-fluorination). Thereaction solution showed a spot of the main product at Rf value of 0.45and multiple spots of by-products at Rf of 0.72-0.76 in silica gel TLC(the TLC was developed with a mixed solvent of CHCl₃ -MeOH, 25:1). Theresulting reaction solution was added with a 5% aqueous solution ofsodium hydrogen carbonate (45 ml). After stirring for 30 minutes, themethylene chloride layer was separated, washed with water and thenconcentrated. The resulting residue was purified by columnchromatography on silica gel in such way that the column was developedwith a developing solvent of CHCl₃ -MeOH (100:1), thereby affording 1.25g (yield: 55%) of the titled Compound (76) as a solid.

(b) Preparation of 5-deoxy-5,5-difluorogentamicin C_(1a) (Compound 77)##STR128##

A solution (about 2.5 ml) in THF of Compound (76) (1.25 g) as obtainedin the step (a) above was added to liquefied ammonia (about 50 ml) at-50° to -60° C. The resulting mixture was added with metal sodium (about400 mg), followed by conducting the reaction for 10 minutes. Thereaction solution showed a spot of the main product at Rf value of 0.70in silica gel TLC (the TLC was developed with the lower layer of a mixeddeveloping solvent of CHCl₃ -MeOH-28% NH₄ OH, 1:1:1). The resultingreaction solution was concentrated and then added with water (10 ml).The resulting mixture was reacted at 80° C. for 3 hours to effect thebreakdown the cyclic carbamate group. The reaction solution showed aspot of the main product at Rf value of 0.36 in silica gel TLC (the TLCwas developed with the lower layer of a mixed developing solvent ofCHCl₃ -MeOH-28% NH₄ OH, 1:1:1).

The resulting reaction solution was neutralized with 6N hydrochloricacid and then adsorbed on the resin "CM-Sephadex C-25 (NH₄ ⁺ -form)".After washing the resin column with water, the column was elutedgradiently with aqueous ammonia, while changing the concentration ofammonia from 0N to 0.2N. Fraction of the eluate containing the desiredcompound were combined together and concentrated under reduced pressure.The resulting residue was purified by column chromatography on silicagel, in such way that the column was developed with a developing solventof CHCl₃ -MeOH-28% NH₄ OH (9:4:1), thereby affording 238 mg (yield: 46%)of the titled Compound (77) as a solid.

[α]_(D) ²⁴ +169° (c 0.4, water)

¹ H-NMR(20% ND₃ /D₂ O): δ1.94(3H, s, 4"-CH₃), 3.27(3H, s, N-CH₃), 5.80and 5.84 (each 1H, d, J_(1'),2' =H_(1"),2" =3.5 Hz)

¹⁹ F-NMR(20% ND₃ /D₂ O): δ-128.15(dt, J_(F),F =246 Hz, J₄,F =J₆,F =21Hz) and -110.35 (dt, J₄,F =J₆,F =4 Hz)

Referential Example 16

(a) Preparation of 1,3,2',6',3"-pentakis(N-benzyloxycarbonyl)netilmicin(Compound 78) ##STR129##

Netilmicin (420 mg) and sodium carbonate (500 mg) were suspended in amixed solvent of water (10 ml) and dioxane (10 ml). The resultingmixture was added with benzyloxycarbonyl chloride (1.0 ml) at 0°-5° C.,followed by conducting the reaction at room temperature for 1 hour. Thereaction solution showed a single spot at Rf value of 0.42 in TLC (theTLC was developed with a mixed solvent of CHCl₃ -MeOH, 25:1). Theresulting reaction solution was concentrated under reduced pressure andthe residue was dissolved in chloroform. The resulting chloroformsolution was washed successively with a 5% aqueous solution of sodiumhydrogen carbonate and water, dried over anhydrous magnesium sulfate andthen concentrated under reduced pressure. The resulting residue waswashed with isopropyl ether to afford 947 mg (yield: 94%) of the titledCompound (78) as a solid.

[α]_(D) ²⁴ +83° (c 0.3 CHCl₃)

(b) Preparation of1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonylnetilmicin(Compound 79) ##STR130##

Compound (78) (1.09 g) as obtained in the step (a) above was dissolvedin DMF (10 ml). The resulting solution was added with sodium hydride(110 mg) at 0°-5° C., followed by conducting the reaction at roomtemperature for 1 hour (for the formation of the cyclic carbamategroup). The resulting reaction solution was added with chloroform (20ml) and water (20 ml) and then neutralized with 1N hydrochloric acid,while stirring. The chloroform layer was separated, washed with waterand then concentrated under reduced pressure. The resulting residue waspurified by column chromatography on silica gel, in such way that thecolumn was developed with a developing solvent of CHCl₃ -MeOH (100:1),thereby affording 931 mg (yield: 94%) of the titled compound (79) as asolid.

[α]_(D) ²⁴ +75° (c 0.3, CHCl₃)

(c) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonylnetilmicin(Compound 80)

Compound (79) (891 mg) as obtained in the step (b) above was dissolvedin pyridine (15 ml), followed by addition of benzoyl chloride (0.5 ml)at 0°-5° C. The reaction was conducted at room temperature for 1 hour(for benzoylation of 2"-hydroxyl group). The resulting reaction solutionwas added with water (0.5 ml) and concentrated under reduced pressure.The resulting residue was dissolved in chloroform and the resultingsolution was washed successively with a 5% aqueous solution of sodiumhydrogen carbonate and water. The chloroform solution was dried overanhydrous magnesium sulfate and then concentrated under reduced pressureto afford 902 mg (yield: 92%) of the titled Compound (80) as solid.

[α]_(D) ²⁴ +94° (c 0.2, CHCl₃)

(d) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carbonyl-5-deoxy-5-oxonetilmicin(Compound 81)

Compound (80) (641 mg) as obtained in the step (c) above was dissolvedin dimethylsufoxide (1.5 ml), followed by addition of acetic anhydride(0.5 ml). The reaction was made at room temperature for 16 hours (forthe oxidation of the 5-hydroxyl group). The resulting reaction solutionwas dissolved in chloroform and the solution in chloroform was washedsuccessiely with water, a 5% aqueous solution of sodium hydrogencarbonate and water. The solution in chloroform was dried over anhydrousmagnesium sulfate and then concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel, in such waythat the column was developed with a developing solvent of CHCl₃ -MeOH(100:1), thereby affording 608 mg (yield: 95%) of the titled Compound(81) as a solid.

[α]_(D) ²⁴ +101° (c 0.2, CHCl₃)

Example 18

(a) Preparation of2"-O-benzoyl-1,3,2',6'-tetrakis(N-benzyloxycarbonyl)-3",4"-N,O-carboyl-5-deoxy-5,5-difluoronetilmicin(Compound 82) ##STR131##

Compound (81) (511 mg) as obtained in Referential Example 16(d) wasdissolved in methylene chloride (8.0 ml). The resulting solution wasadded with diethylaminosulfur trifluoride (0.6 ml) under ice-cooling,followed by conducting the reaction at room temperature for 6 hours (forthe di-fluorination). The resulting reaction solution was added with a5% aqueous solution of sodium hydrogen carbonate (15 ml) and thenstirred for 30 minutes. The methylene chloride layer was separated,washed with water and then concentrated. The residue was purified bycolumn chromatography on silica gel, in such way that the column wasdeveloped with a developing solvent of CHCl₃ -MeOH (100:1), therebyaffording 325 mg (yield: 62%) of the titled Compound (82) as a solid.

[α]_(D) ²⁴ +90° (c 0.3, CHCl₃)

(b) Preparation of 5-deoxy-5,5-difluoronetilmicin (Compound 83)##STR132##

A solution (about 1 ml) in THF of Compound (82) (245 mg) was added toliquefied ammonia (about 25 ml) at -50° to -60° C. The resultingsolution was added with metal sodium (about 300 mg), followed byconducting the reaction for 10 minutes. The resulting reaction solutionwas added with methanol (1 ml), concentrated, and then added with water(5 ml). The resulting mixture was reacted at 80° C. for 3 hours (for thebreakdown of the cyclic carbamate group). The reaction solution showed aspot of the main product at Rf value of 0.25 in silica gel TLC (the TLCwas developed with CHCl₃ -MeOH-28% NH₄ OH, 9:4:1).

The resulting reaction solution was neutalized with 6N hydrochloric acidand then adsorbed on the resin "CM-Sephadex C-25 (NH₄ ⁺ -form)". Afterwashing the resin column with water, the column was eluted gradientlywith aqueous ammonia, while changing the concentration of ammonia from0N to 0.2N. Fractions of the eluate containing the desired compound werecollected, combined together and concentrated under reduced pressure.The resulting residue was purified by column chromatography on silicagel, in such way that the column was developed with a developing solventof CHCl₃ -MeOH-28% NH₄ OH (9:4:1), thereby affording 54.4 mg (yield:52%) of the titled Compound (83) as a solid.

[α]_(D) ²⁴ +148° (c 1.0, water)

¹⁹ F-NMR(20% ND₃ /D₂ O: δ-128.68(dt, J_(F),F =244 Hz, J₄,F =J₆,F =20 Hz)and -113.50(d)

Example 19

Preparation of 5-deoxy-5,5-difluoroneamine (Compound 84) ##STR133##

Monocarbonate of 5-deoxy-5,5-difluorokanamycin B (which is Compound(23)) obtained in Example 5(c) (625 mg) was dissolved in an 4N aqueoushydrochloric acid solution (13 ml). Subsequently, the reaction wasconducted at 100° C. for 1 hour to hydrolyze the Compound (23).

The resulting reaction solution was concentrated under reduced pressureand then neutralized with aqueous ammonia. The neutralized solution waspassed through a column of the resin "Amberlite CG-50" (NH₄ ⁺ -form) onwhich the reaction product was adsorbed. The resin column was elutedgradiently with aqueous ammonia, while changing the concentration ofammonia 0N to 0.2N. Fractions of the eluate containing the intendedcompound were collected, combined together, and concentrated to drynessto afford 251 mg (yield: 52%, calculated as themonocarbonate-monohydrate) of the titled Compound (84) as a solid.

[α]_(D) ²⁰ +70° (c 1, water)

Example 20

Preparation of 5,3',4'-trideoxy-5,5-difluoroneamine (Compound 85)##STR134##

5,3',4'-Trideoxy-5,5-difluorokanamycin B, namely Compound (40) asobtained in Example 9(c) (10.0 g) is dissolved in a 4N aqueoushydrochloric acid solution (200 ml). The reaction was conducted at 100°C. for 1 hour to hydrolyze Compound (40).

The resulting reaction solution was concentrated under reduced pressureand then neutralized with aqueous ammonia. The neutralized solution waspassed through a column of the resin "Amberlite CG-50" (NH₄ ⁺ -form)(850 ml) on which the reaction product was adsorbed. The resin columnwas washed with water and then eluted gradiently with 0.2N aqueousammonia. Fractions of the eluate containing the intended compound werecollected, combined together and then concentrated to afford 4.72 g(yield: 72%) of the titled Compound (85) as a solid.

[α]_(D) ²⁰ +790 (c 1.0, water)

INDUSTRIAL APPLICABILITY

5-Deoxy-5,5-difluoro derivatives of the aminoglycosidic antibiotics andtheir 1-N-(α-hydroxy-ω-aminoalkanoyl)-derivatives according to thisinvention exhibit high antibacterial activity against bacteria sensitiveto aminoglycosidic antibiotics and against bacteria resistant toaminoglycosidic antibiotics, and also, they show significantly reducedtoxicity, so that they are useful as antibacterial agent for therepeutictreatment of bacterial infections.

We claim:
 1. A 4-O-(aminoglycosyl)- or 4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamine derivative represented by theformula ##STR135## wherein R¹ is a hydrogen atom, an alkyl group of 1-4carbon atoms or an α-hydroxy-ω-aminoalkanoyl group of the formula##STR136## where n is an integer of 1 to 3, G¹ is either anaminoglycosyl group of the formula ##STR137## where A¹ is a hydroxylgroup or amino group, A² and A³ are independently a hydrogen atom,hydroxyl group or fluoro group, A⁴ is a hydrogen atom or methyl group,and A⁵ is a hydrogen atom or methyl group, or G² is a4'-eno-aminoglycosyl group of the formula ##STR138## where A¹, A², A⁴and A⁵ independently have the same meanings as defined above, and G² iseither a hydrogen atom, or a 3"-amino-3"-deoxyglycosyl group of theformula ##STR139## where B¹ is a hydrogen atom or methyl group, B² andB³ are independently a hydrogen atom, hydroxyl group or methyl group,and B⁴ is a hydrogen atom or a hydroxymethyl group (--CH₂ OH), or G² isa 2"-amino-2"-deoxyglycosyl group of the formula ##STR140## where B¹,B², B³ and B⁴ independently have the same meanings as defined above, ora pharmaceutically acceptable acid addition salt of said derivative. 2.A streptamine derivative as claimed in claim 1, which is a4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptamine derivativerepresented by the formula ##STR141## wherein A¹, A², A³, A⁴ and A⁵, aswell as B¹, B², B³ and B⁴ have the same meanings as defined for theformula (I) described in claim 1, and which is such a compound belongingto a derivative of kanamycin A, a derivative of kanamycin B, aderivative of gentamicin C₁, a derivative of gentamicin C_(1a) or aderivative of gentamicin C₂ or a derivative of sagamicin.
 3. Astreptamine derivative as claimed in claim 1, which is a compoundrepresented by the formula ##STR142## wherein (i) A² and A³ are each ahydroxyl group, or (ii) A² is a hydrogen atom and A³ is a hydroxylgroup, or (iii) A² and A³ are each a hydrogen atom, and which is namely(i) 5-deoxy-5,5-difluorokanamycin A, or (ii)5,3'-dideoxy-5,5-difluorokanamycin A, or (iii)5,3',4'-trideoxy-5,5-difluorokanamycin A.
 4. A streptamine derivative asclaimed in claim 1, which is a compound represented by the formula##STR143## wherein (i) A² and A³ are each a hydroxyl group, or (ii) A²is a hydrogen atom and A³ is a hydroxyl group, or (iii) A² and A³ areeach a hydrogen atom, or (iv) A² is a fluoro group and A³ is a hydroxylgroup, or (v) A² is a fluoro group and A³ is a hydrogen atom, and whichis namely (i) 5-deoxy-5,5-difluorokanamycin B, or (ii)5,3'-dideoxy-5,5-difluorokanamycin B, or (iii)5,3',4'-trideoxy-5,5-difluorokanamycin B, or (iv)5,3'-dideoxy-5,5,3'-trifluorokanamycin B, or (v)5,3',4'-trideoxy-5,5,3'-trifluorokanamycin B.
 5. A streptaminederivative as claimed in claim 1, which is a compound represented by theformula ##STR144## wherein (i) A² and A³ are each a hydrogen atom and A⁴and A⁵ are each a methyl group, or (ii) A², A³, A⁴ and A⁵ are all andeach a hydrogen atom, or (iii) A², A³ and A⁵ are all and each a hydrogenatom and A⁴ is methyl group, or (iv) A², A³ and A⁴ are all and each ahydrogen atom and A⁵ is methyl group, and which is namely (i)5-deoxy-5,5-difluorogentamicin C₁, or (ii)5-deoxy-5,5-difluorogentamicin C_(1a), or (iii)5-deoxy-5,5-difluorogentamicin C₂, or (iv)5-deoxy-5,5-difluorosagamicin.
 6. A streptamine derivative as claimed inclaim 1, which is a compound represented by the formula ##STR145##wherein (i) R¹ is a hydrogen atom and A², A⁴ and A⁵ are all and each ahydrogen atom, or (ii) R¹ is ethyl group and A², A⁴ and A⁵ are all andeach a hydrogen atom, and which is namely (i)5-deoxy-5,5-difluorosisomicin or (ii) 5-deoxy-5,5-difluoronetilmicin. 7.A streptamine derivative as claimed in claim 1, which is a1-N-(α-hydroxy-ω-aminoalkanoyl)-4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptaminederivative represented by the formula ##STR146## wherein n is an integerof 1 to 3, and A¹, A², A³, A⁴ and A⁵ as well as B¹, B², B³ and B⁴respectively have the same meanings as defined for the formula (I)described in claim
 1. 8. A streptamine derivative as claimed in claim 1,which is a compound represented by the formula ##STR147## wherein n isan integer of 1 to 3, and (i) A² and A³ are each a hydroxyl group, or(ii) A² is a hydrogen atom and A³ is hydroxyl group, or (iii) A² and A³are each a hydrogen atom, and which is namely a1-N-(α-hydroxy-ω-aminoalkanoyl) derivative of anyone of (i)5-deoxy-5,5-difluorokanamycin A or (ii)5,3'-dideoxy-5,5-difluorokanamycin A or (iii)5,3',4'-trideoxy-5,5-difluorokanamycin A.
 9. A streptamine derivative asclaimed in claim 1, which is a compound represented by the formula##STR148## wherein n is an integer of 1 to 3, and (i) A² and A³ are eacha hydroxyl group, or (ii) A² is a hydrogen atom and A³ is hydroxylgroup, or (iii) A² and A³ are each a hydrogen atom, or (iv) A² is afluoro group and A³ is hydroxyl group, or (v) A² is a fluoro group andA³ is a hydrogen atom, and which is namely a1-N-(α-hydroxy-ω-aminoalkanoyl) derivative of anyone of (i)5-deoxy-5,5-difluorokanamycin B or (ii)5,3'-dideoxy-5,5-difluorokanamycin B or (iii)5,3',4'-trideoxy-5,5-difluorokanamycin B or (iv)5,3'-dideoxy-5,5,3'-trifluorokanamycin B or (v)5,3',4'-trideoxy-5,5,3'-trifluorokanamycin B.
 10. A streptaminederivative as claimed in claim 1, which is a compound represented by theformula ##STR149## wherein n is an integer of 1 to 3, and (i) A² and A³are each a hydrogen atom and A⁴ and A⁵ are each a methyl group, or (ii)A², A³, A⁴ and A⁵ are all and each a hydrogen atom, or (iii) A², A³ andA⁵ are all and each a hydrogen atom and A⁴ is methyl group, or (iv) A²,A³ and A⁴ are all and each a hydrogen atom and A⁵ is methyl group, andwhich is a 1-N-(α-hydroxy-ω-aminoalkanoyl) derivative of anyone of (i)5-deoxy-5,5-difluorogentamicin C₁ or (ii) 5-deoxy-5,5-difluorogentamicinC_(1a) or (iii) 5-deoxy-5,5-difluorogentamicin C₂ or (iv)5-deoxy-5,5-difluorosagamicin.
 11. A streptamine derivative as claimedin claim 1, which is 5-deoxy-5,5-difluoroseldomycin factor 3 representedby the formula ##STR150##
 12. An antibacterial composition,characterized in that said composition comprises a 4-O-(aminoglycosyl)-or 4,6-di-O-(aminoglycosyl)-2,5-dideoxy-5,5-difluorostreptaminederivative represented by the formula ##STR151## wherein R¹, G¹ and G²respectively have the same meanings as defined for the formula (I)described in claim 1, as the active ingredient in an antibacteriallyeffective amount, in association with a pharmaceutically acceptablecarrier for the active ingredient.